Methods in Bioengineering: Alternative Technologies to Animal Testing (The Artech House Methods in Bioengineering)

298 Pages·2010·0.28 MB·English

by  Tim Maguire

Methods in Bioengineering
Alternative Technologies to Animal Testing
The Artech House Methods in Bioengineering Series
Series Editors-in-Chief
Martin L.Yarmush, M.D., Ph.D.
Robert S.Langer, Sc.D.
Methods in Bioengineering: Alternative Technologies to Animal Testing,
Tim Maguire and Eric Novik, editors
Methods in Bioengineering: Biomicrofabrication andBiomicrofluidics,
Jeffrey D.Zahn, editor
Methods in Bioengineering: Microdevices in Biology and Medicine,
YaakovNahmias andSangeeta N.Bhatia, editors
Methods in Bioengineering: Nanoscale Bioengineering and Nanomedicine,
KaushalRegeand IgorMedintz, editors
Methods in Bioengineering: Stem Cell Bioengineering,
BijuParekkadanand Martin L. Yarmush, editors
Methods in Bioengineering: Systems Analysis of Biological Networks,
ArulJayaraman andJuergenHahn, editors
Methods in Bioengineering
Alternative Technologies to Animal Testing
Tim Maguire
Rutgers University
EricNovik
HurelCorporation
Editors
artechhouse.com
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the U. S. Library of Congress.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library.
ISBN-13: 978-1-60807-011-4
Cover design by Vicki Kane
© 2010ArtechHouse. All rights reserved.
Printed and bound in the United States of America. No part of this book may be reproduced or
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All terms mentioned in this book that are known to be trademarks or service marks have been
appropriately capitalized.ArtechHouse cannot attest to the accuracy of this information. Use of
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mark.
10 9 8 7 6 5 4 3 2 1
Contents
Preface xiii
CHAPTER 1
Current Methods for Prediction of Human Hepatic Clearance Using In Vitro
Intrinsic Clearance 1
1.1 Introduction 2
1.2 Materials 3
1.3 Methods 3
1.3.1 Thawing thehepatocytes 3
1.3.2 Clearance study using ahepatocytesuspension 3
1.3.3 Clearance study using a platedhepatocyteculture 4
1.3.4 Clearance study using a platedhepatocyteculture under a flow
condition 4
1.3.5 Sampling for the clearance study 6
1.3.6 Sample analysis using LC-MS/MS 6
1.4 Data Acquisition, Anticipated Results, and Interpretation 7
1.4.1 Hepatocytesuspension and platedhepatocytesystem 7
1.4.2 Physiologically basedmicrofluidicsystems 8
1.5 Discussion and Commentary 8
1.5.1 Hepatocytesuspension system 8
1.5.2 Platedhepatocytesystem 11
1.5.3 Physiologically basedmicrofluidicsystems 12
1.6 Summary 13
References 15
CHAPTER 2
Use of Permeability from Cultured Cell Lines and PAMPA System and Absorption
from Experimental Animals for the Prediction of Absorption in Humans 19
2.1 Introduction 20
2.2 Materials 21
2.3 Methods 21
2.3.1 Cultured cell system 21
2.3.2 PAMPA system 24
2.3.3 In vivo absorption measurements 25
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Contents
2.4 Data Acquisition, Anticipated Results, and Interpretation 25
2.4.1 Data analysis 25
2.4.2 Results and interpretation 26
2.5 Discussion and Commentary 30
2.5.1 Cell culture and PAMPA systems 30
2.5.2 Absorption in experimental animals 35
2.5.3 Rats 35
2.5.4 Dogs 36
2.5.5 Monkeys 37
2.6 Summary 38
References 38
CHAPTER 3
Aggregating Brain Cell Cultures forNeurotoxicityTests 41
3.1 Introduction 42
3.2 Experimental Design 43
3.3 Materials 44
3.3.1 Animals 44
3.3.2 Special equipment 45
3.3.3 Reagents 46
3.3.4 Preparation of solutions and media 47
3.4 Methods 49
3.4.1 Washing and sterilizing the glassware 49
3.4.2 Cell isolation and culture preparation 49
3.4.3 Maintenance of aggregating brain cell cultures (media
replenishment and subdivision) 51
3.4.4 Preparation and treatment of replicate cultures 52
3.4.5 Harvest of replicate cultures for various analytical procedures 53
3.4.6 Examples of sample preparation and use for various analytical
procedures 53
3.4.7 Data Analysis 54
3.5 Anticipated Results 55
3.6 Discussion and Commentary 57
3.7 Application Notes 57
3.8 Summary Points 58
Acknowledgments 59
References 59
CHAPTER4
ApproachesTowardsaMultiscaleModelofSystemicInflammationinHumans 61
4.1 Introduction 62
4.2 Materials 64
4.2.1 Humanendotoxinmodel and data collection 64
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Contents
4.3 Methods 65
4.3.1 Transcriptionaldynamics and intrinsic responses 65
4.3.2 Modeling inflammation at the cellular level 67
4.3.3 Modeling inflammation at the systemic level 73
4.4 Results 77
4.4.1 Elements of themultiscalehost response model of human
inflammation 77
4.4.2 Estimation of relevant model parameters 77
4.4.3 Qualitative assessment of the model 80
4.5 Conclusions 91
Acknowledgments 91
References 92
CHAPTER 5
ALiposomeAssay for Evaluating the Ocular Toxicity of Chemicals 99
5.1 Introduction 100
5.2 Experimental Design 101
5.3 Materials 102
5.4 Methods 103
5.4.1 Preparation ofcalcein-loadedliposomes 103
5.4.2 Separation of bulkcalceinfrom loadedliposomeswith
Sephadex 104
5.4.3 Ocular toxicity experiments using dye-loadedliposomes 106
5.5 Data Acquisition, Anticipated Results, and Interpretation 108
5.6 Discussion and Commentary 109
5.7 Application Notes 111
5.8 Summary Points 112
References 113
CHAPTER 6
Prediction of Potential DrugMyelotoxicityby In Vitro Assays onHematopoietic
Progenitors 115
6.1 Introduction 116
6.2 Experimental Design 116
6.3 Materials 117
6.3.1 Reagents 118
6.4 Methods 118
6.4.1 Preparation ofmethylcellulosestocks 118
6.4.2 Source ofmurinehematopoieticprogenitors 118
6.4.3 Source of humanhematopoieticprogenitors 119
6.4.4 Technical procedure forGM-CFUtest 121
6.4.5 Passing from screening phase to IC determination phase 122
6.4.6 Incubator humidity test 122
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Contents
6.4.7 Scoring the colonies 123
6.4.8 Criteria for colony counting 123
6.5 Data Acquisition, Anticipated Results, and Interpretation 124
6.5.1 Statistical guidelines 125
6.6 Discussion and Commentary 126
6.7 Application Notes 128
6.8 Summary Points 128
Acknowledgments 130
References 130
CHAPTER 7
EpigeneticallyStabilized PrimaryHepatocyteCultures: A Potential Sensitive
Screening Tool forNongenotoxicCarcinogenicity 133
7.1 Introduction 134
7.2 Experimental Design 135
7.3 Materials 135
7.3.1 Reagents 135
7.3.2 Facilities/Equipment 139
7.4 Methods 139
7.4.1 Isolation ofhepatocytesfrom rat liver 139
7.4.2 Cultivation of primary rathepatocytes(Troubleshooting Table) 141
7.5 Data Acquisition 141
7.6 Anticipated Results and Interpretation 141
7.7 Discussion and Commentary 143
7.8 Application Notes 144
7.9 Summary Points 144
Acknowledgements 145
References 145
CHAPTER 8
A Statistical Method to Reduce In Vivo Product Testing Using Related In Vitro
Tests and ROC Analysis 147
8.1 Introduction 148
8.2 Experimental Design 149
8.3 Materials 149
8.4 Methods 150
8.4.1 Step-by-step protocol for the analysis of data usingAnalyse-It 152
8.5 Results 154
8.6 Discussion and Commentary 154
8.6.1 Selecting the proper secondary test 154
8.6.2 Determining the sample size for calibration andrecalibration 155
8.6.3 Regulatory concerns 156
8.6.4 Determining the frequency ofrecalibration 156
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8.6.5 Determining the need for confirmatory testing 157
8.6.6 Statistical analysis 157
8.7 Summary Points 158
Acknowledgments 158
References 158
CHAPTER 9
Application of the Benchmark Approach in the Correlation of In Vitro and
In Vivo Data in Developmental Toxicity 159
9.1 Introduction 160
9.2 Materials and Methods 162
9.2.1 Derivation of in vitroBMCandBMDvalues 163
9.2.2 In vitro–in vivo correlation 164
9.3 Discussion and Commentary 167
References 168
CHAPTER 10
Three-Dimensional Cell Culture of Canine Uterine Glands 171
10.1 Introduction 172
10.2 Materials 173
10.2.1 Cell culture 173
10.2.2 Histologicalpreparation for light microscopy 173
10.2.3 Histologicalpreparation for electron microscopy 174
10.3 Methods 174
10.3.1 Cell culture 174
10.3.2 Histologicalpreparation for light microscopy 176
10.3.3 Histologicalpreparation for electron microscopy 177
10.3.4 Imaging 178
10.4 Anticipated Results 178
10.5 Discussion and Commentary 178
10.6 Application Notes 180
10.7 Summary Points 181
References 181
CHAPTER 11
Markers for an In Vitro Skin Substitute 183
11.1 Introduction 184
11.2 Experimental Design 185
11.3 Materials 185
11.3.1 Human tissue-engineered skin substitute reconstructed
by the self-assembly approach 185
11.4 Methods 188
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