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Publisher Summary 1
Artech House Methods in Bioengineering Series
Series editors in chief Martin L. Yarmush, Massachusetts General Hospital/Harvard Medical School, and Robert S. Langer, Harvard-MIT Division of Health Science and Technology
Written and edited by recognized experts in the field, the Artech House Methods in Bioengineering Series offers detailed guidance on innovative methods for addressing specific bioengineering laboratory challenges. Offering highly practical and fully illustrated presentations of each topic, these books provide research engineers, scientists, and students with step-by-step procedures, clear examples, and effective ways to overcome problems that may be encountered.
Providing alternatives to animal testing is one of the hottest topics in biomedical research, and this groundbreaking volume addresses this critical issue head on. This unique book presents techniques and methods at the forefront of scientific research that have the potential to replace certain whole animal tests. Moreover, this book provides a platform where other widely accepted techniques and scientific advancements can be collated into a concise set of methods that can be implemented within both academic and industrial communities. Professionals and researchers find guidance in critical methods, including:
-Prediction of human hepatic clearance using in vitro intrinsic clearance;
-Approaches towards a multiscale model of systemic inflammation in humans;
-A liposome assay for evaluating the ocular toxicity of chemicals;
-The application of the benchmark approach in the correlation of in vitro and in vivo data in developmental toxicity;
-A 3D model of the human epithelial airway barrier;
-Experimental wear assessment of tibial inserts for total knee replacement.
Tim Maguire works at a startup company, where he is pursuing the creation of in vitro drug screening systems, integrating optimized human hepatocyte cultures with microfluidic systems. He holds a B.S. in a chemical engineering and a Ph.D. in biomedical engineering, both from Rutgers University.
Eric Novik works at a startup company and is involved in all aspects of development and commercialization of patented microfluidic, cell-based platforms for use in drug discovery and development, consumer and industrial product testing, and related fields. He holds a B.S. and a Ph.D. in biomedical engineering from Rutgers University.
Publisher Summary 2
This volume is comprised of 15 scientific papers considering different laboratory alternatives to animal testing. Each includes an abstract, introduction, a section on materials and methods, data acquisition, anticipated results and interpretation, discussion, commentary, and concluding notes as well as numerous equations, tables, and graphs. Topics include aggregating brain cell cultures for neurotoxicity tests, markers for an in vitro skin substitute, alternatives for absorption testing, and a liposome assay for evaluating the ocular toxicity of chemicals. Contributors are researchers associated with universities and pharmaceutical companies around the world. Annotation 漏2010 Book News, Inc., Portland, OR (booknews.com)
目录
Table Of Contents:
Preface xiii
Chapter 1 Current Methods for Prediction of Human Hepatic Clearance Using In Vitro Intrinsic Clearance 1(18)
1.1 Introduction 2(1)
1.2 Materials 3(1)
1.3 Methods 3(4)
1.3.1 Thawing the hepatocytes 3(1)
1.3.2 Clearance study using a hepatocyte suspension 3(1)
1.3.3 Clearance study using a plated hepatocyte culture 4(1)
1.3.4 Clearance study using a plated hepatocyte culture under a flow condition 4(2)
1.3.5 Sampling for the clearance study 6(1)
1.3.6 Sample analysis using LC-MS/MS 6(1)
1.4 Data Acquisition, Anticipated Results, and Interpretation 7(1)
1.4.1 Hepatocyte suspension and plated hepatocyte system 7(1)
1.4.2 Physiologically based microfluidic systems 8(1)
1.5 Discussion and Commentary 8(5)
1.5.1 Hepatocyte suspension system 8(3)
1.5.2 Plated hepatocyte system 11(1)
1.5.3 Physiologically based microfluidic systems 12(1)
1.6 Summary 13(2)
References 15(4)
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(22)
2.1 Introduction 20(1)
2.2 Materials 21(1)
2.3 Methods 21(4)
2.3.1 Cultured cell system 21(3)
2.3.2 PAMPA system 24(1)
2.3.3 In vivo absorption measurements 25(1)
2.4 Data Acquisition, Anticipated Results, and Interpretation 25(5)
2.4.1 Data analysis 25(1)
2.4.2 Results and interpretation 26(4)
2.5 Discussion and Commentary 30(8)
2.5.1 Cell culture and PAMPA systems 30(5)
2.5.2 Absorption in experimental animals 35(1)
2.5.3 Rats 35(1)
2.5.4 Dogs 36(1)
2.5.5 Monkeys 37(1)
2.6 Summary 38(1)
References 38(3)
Chapter 3 Aggregating Brain Cell Cultures for Neurotoxicity Tests 41(20)
3.1 Introduction 42(1)
3.2 Experimental Design 43(1)
3.3 Materials 44(5)
3.3.1 Animals 44(1)
3.3.2 Special equipment 45(1)
3.3.3 Reagents 46(1)
3.3.4 Preparation of solutions and media 47(2)
3.4 Methods 49(6)
3.4.1 Washing and sterilizing the glassware 49(1)
3.4.2 Cell isolation and culture preparation 49(2)
3.4.3 Maintenance of aggregating brain cell cultures (media replenishment and subdivision) 51(1)
3.4.4 Preparation and treatment of replicate cultures 52(1)
3.4.5 Harvest of replicate cultures for various analytical procedures 53(1)
3.4.6 Examples of sample preparation and use for various analytical procedures 53(1)
3.4.7 Data Analysis 54(1)
3.5 Anticipated Results 55(2)
3.6 Discussion and Commentary 57(1)
3.7 Application Notes 57(1)
3.8 Summary Points 58(1)
Acknowledgments 59(1)
References 59(2)
Chapter 4 Approaches Towards a Multiscale Model of Systemic Inflammation in Humans 61(38)
4.1 Introduction 62(2)
4.2 Materials 64(1)
4.2.1 Human endotoxin model and data collection 64(1)
4.3 Methods 65(12)
4.3.1 Transcriptional dynamics and intrinsic responses 65(2)
4.3.2 Modeling inflammation at the cellular level 67(6)
4.3.3 Modeling inflammation at the systemic level 73(4)
4.4 Results 77(14)
4.4.1 Elements of the multiscale host response model of human inflammation 77(1)
4.4.2 Estimation of relevant model parameters 77(3)
4.4.3 Qualitative assessment of the model 80(11)
4.5 Conclusions 91(1)
Acknowledgments 91(1)
References 92(7)
Chapter 5 A Liposome Assay for Evaluating the Ocular Toxicity of Chemicals 99(16)
5.1 Introduction 100(1)
5.2 Experimental Design 101(1)
5.3 Materials 102(1)
5.4 Methods 103(5)
5.4.1 Preparation of calcein-loaded liposomes 103(1)
5.4.2 Separation of bulk calcein from loaded liposomes with Sephadex 104(2)
5.4.3 Ocular toxicity experiments using dye-loaded liposomes 106(2)
5.5 Data Acquisition, Anticipated Results, and Interpretation 108(1)
5.6 Discussion and Commentary 109(2)
5.7 Application Notes 111(1)
5.8 Summary Points 112(1)
References 113(2)
Chapter 6 Prediction of Potential Drug Myelotoxicity by In Vitro Assays on Hematopoietic Progenitors 115(18)
6.1 Introduction 116(1)
6.2 Experimental Design 116(1)
6.3 Materials 117(1)
6.3.1 Reagents 118(1)
6.4 Methods 118(6)
6.4.1 Preparation of methylcellulose stocks 118(1)
6.4.2 Source of murine hematopoietic progenitors 118(1)
6.4.3 Source of human hematopoietic progenitors 119(2)
6.4.4 Technical procedure for GM-CFU test 121(1)
6.4.5 Passing from screening phase to IC determination phase 122(1)
6.4.6 Incubator humidity test 122(1)
6.4.7 Scoring the colonies 123(1)
6.4.8 Criteria for colony counting 123(1)
6.5 Data Acquisition, Anticipated Results, and Interpretation 124(2)
6.5.1 Statistical guidelines 125(1)
6.6 Discussion and Commentary 126(2)
6.7 Application Notes 128(1)
6.8 Summary Points 128(2)
Acknowledgments 130(1)
References 130(3)
Chapter 7 Epigenetically Stabilized Primary Hepatocyte Cultures: A Potential Sensitive Screening Tool for Nongenotoxic Carcinogenicity 133(14)
7.1 Introduction 134(1)
7.2 Experimental Design 135(1)
7.3 Materials 135(4)
7.3.1 Reagents 135(4)
7.3.2 Facilities/Equipment 139(1)
7.4 Methods 139(2)
7.4.1 Isolation of hepatocytes from rat liver 139(2)
7.4.2 Cultivation of primary rat hepatocytes (Troubleshooting Table) 141(1)
7.5 Data Acquisition 141(1)
7.6 Anticipated Results and Interpretation 141(2)
7.7 Discussion and Commentary 143(1)
7.8 Application Notes 144(1)
7.9 Summary Points 144(1)
Acknowledgements 145(1)
References 145(2)
Chapter 8 A Statistical Method to Reduce In Vivo Product Testing Using Related In Vitro Tests and ROC Analysis 147(12)
8.1 Introduction 148(1)
8.2 Experimental Design 149(1)
8.3 Materials 149(1)
8.4 Methods 150(4)
8.4.1 Step-by-step protocol for the analysis of data using Analyse-It 152(2)
8.5 Results 154(1)
8.6 Discussion and Commentary 154(4)
8.6.1 Selecting the proper secondary test 154(1)
8.6.2 Determining the sample size for calibration and recalibration 155(1)
8.6.3 Regulatory concerns 156(1)
8.6.4 Determining the frequency of recalibration 156(1)
8.6.5 Determining the need for confirmatory testing 157(1)
8.6.6 Statistical analysis 157(1)
8.7 Summary Points 158(1)
Acknowledgments 158(1)
References 158(1)
Chapter 9 Application of the Benchmark Approach in the Correlation of In Vitro and In Vivo Data in Developmental Toxicity 159(12)
9.1 Introduction 160(2)
9.2 Materials and Methods 162(5)
9.2.1 Derivation of in vitro BMC and BMD values 163(1)
9.2.2 In vitro-in vivo correlation 164(3)
9.3 Discussion and Commentary 167(1)
References 168(3)
Chapter 10 Three-Dimensional Cell Culture of Canine Uterine Glands 171(12)
10.1 Introduction 172(1)
10.2 Materials 173(1)
10.2.1 Cell culture 173(1)
10.2.2 Histological preparation for light microscopy 173(1)
10.2.3 Histological preparation for electron microscopy 174(1)
10.3 Methods 174(4)
10.3.1 Cell culture 174(2)
10.3.2 Histological preparation for light microscopy 176(1)
10.3.3 Histological preparation for electron microscopy 177(1)
10.3.4 Imaging 178(1)
10.4 Anticipated Results 178(1)
10.5 Discussion and Commentary 178(2)
10.6 Application Notes 180(1)
10.7 Summary Points 181(1)
References 181(2)
Chapter 11 Markers for an In Vitro Skin Substitute 183(22)
11.1 Introduction 184(1)
11.2 Experimental Design 185(1)
11.3 Materials 185(3)
11.3.1 Human tissue-engineered skin substitute reconstructed by the self-assembly approach 185(3)
11.4 Methods 188(6)
11.4.1 Preparation of solutions and materials for the in vitro fabrication of human skin substitutes by the self-assembly approach 188(1)
11.4.2 In vitro fabrication of human skin substitutes by the self-assembly approach 189(2)
11.4.3 Tissue preservation and sectioning 191(1)
11.4.4 Preparation of solutions and materials for immunofluorescence 192(1)
11.4.5 Immunofluorescent labeling of human skin substitutes 192(1)
11.4.6 Histological analysis 193(1)
11.4.7 Transmission electron microscopy 193(1)
11.4.8 Statistical analysis 193(1)
11.5 Anticipated Results 194(4)
11.6 Discussion and Commentary 198(1)
11.7 Application Notes 199(1)
11.8 Summary Points 200(1)
Acknowledgments 201(1)
References 201(4)
Chapter 12 3D Culture of Primary Chondrocytes, Cartilage, and Bone/Cartilage Explants in Simulated Microgravity 205(22)
12.1 Introduction 206(2)
12.2 Experimental Design 208(3)
12.2.1 Culture models 208(1)
12.2.2 The RCCS bioreactor and its operational conditions 208(1)
12.2.3 Animals 209(2)
12.3 Materials 211(1)
12.3.1 Equipment for cell/tissue culture and preparation of samples 211(1)
12.3.2 Chemicals 211(1)
12.4 Methods 212(6)
12.4.1 Preparation of tissue explants 212(1)
12.4.2 Isolation of chondrocytes 212(2)
12.4.3 2D culture of isolated chondrocytes (traditional monolayer in static fluid conditions) 214(1)
12.4.4 3D culture of isolated chondrocytes (homotypic aggregates) 214(2)
12.4.5 3D culture of fragments of articular cartilage explants 216(1)
12.4.6 3D culture of undissected, complete proximal tibial epiphyses 217(1)
12.4.7 Histomorphological study of chondrocytes and cartilage tissue 217(1)
12.5 Anticipated Results 218(2)
12.6 Discussion 220(2)
12.6.1 Discussion of pitfalls 220(1)
12.6.2 General discussion and commentary 221(1)
12.7 Application Notes 222(1)
12.8 Summary Points 223(1)
Acknowledgments 224(1)
References 224(3)
Chapter 13 Alternatives for Absorption Testing 227(12)
13.1 Introduction 228(1)
13.2 Materials 229(1)
13.2.1 Franz diffusion cell 229(1)
13.2.2 Consumables 229(1)
13.2.3 Chemicals and solutions 229(1)
13.2.4 Technical equipment 230(1)
13.3 Methods 230(4)
13.3.1 Skin preparation 230(1)
13.3.2 Determination of skin penetration using the Franz cell setup 230(1)
13.3.3 Determination of skin permeation using the Franz cell setup 231(1)
13.3.4 Skin absorption studies with commercially available 3D skin models 231(1)
13.3.5 Quality control 231(1)
13.3.6 Data evaluation 232(1)
13.3.7 Biostatistics 233(1)
13.4 Results and Discussion 234(2)
13.5 Discussion of Pitfalls and Troubleshooting 236(1)
13.6 Summary 236(1)
References 237(2)
Chapter 14 A 3D Model of the Human Epithelial Airway Barrier 239(22)
14.1 Introduction 240(1)
14.2 Experimental Design 241(1)
14.3 Materials 241(2)
14.3.1 General materials 241(1)
14.3.2 Epithelial cell cultures鈥攖hawing 241(1)
14.3.3 Epithelial cell cultures鈥攃ulturing 241(1)
14.3.4 Isolation of monocyte-derived macrophages (MDM) and dendritic cells (MDDQ 242(1)
14.3.5 Triple cell coculture 242(1)
14.3.6 Transepithelial electrical resistance (TEER) measurements 242(1)
14.3.7 Staining for laser scanning microscopy (LSM) 243(1)
14.3.8 Embedding for transmission electron microscopy (TEM) 243(1)
14.4 Methods 243(10)
14.4.1 Epithelial cells 243(3)
14.4.2 Isolation of monocyte-derived macrophages (MDM) and dendritic cells (MDDC) 246(3)
14.4.3 TEER measurements 249(1)
14.4.4 Staining for LSM 250(1)
14.4.5 Fixation and embedding of cells for transmission electron microscopy (TEM) 251(2)
14.5 Anticipated Results 253(4)
14.6 Discussion and Commentary 257(1)
14.7 Application Notes 257(1)
14.8 Summary Points 257(1)
Acknowledgments 257(1)
References 258(3)
Chapter 15 Experimental Wear Assessment of Tibial Inserts for Total Knee Replacement 261(12)
15.1 Introduction 262(1)
15.2 Experimental Design 263(1)
15.3 Materials 263(2)
15.4 Methods 265(3)
15.4.1 Management of the specimens 265(1)
15.4.2 Wear test procedure 266(1)
15.4.3 Examination of worn tibial inserts surfaces 267(1)
15.5 Anticipated Results 268(1)
15.6 Discussion and Commentary 269(1)
15.7 Application Notes 269(1)
15.8 Summary Points 270(1)
Acknowledgments 271(1)
References 271(2)
About the Editors 273(2)
Index 275
Preface xiii
Chapter 1 Current Methods for Prediction of Human Hepatic Clearance Using In Vitro Intrinsic Clearance 1(18)
1.1 Introduction 2(1)
1.2 Materials 3(1)
1.3 Methods 3(4)
1.3.1 Thawing the hepatocytes 3(1)
1.3.2 Clearance study using a hepatocyte suspension 3(1)
1.3.3 Clearance study using a plated hepatocyte culture 4(1)
1.3.4 Clearance study using a plated hepatocyte culture under a flow condition 4(2)
1.3.5 Sampling for the clearance study 6(1)
1.3.6 Sample analysis using LC-MS/MS 6(1)
1.4 Data Acquisition, Anticipated Results, and Interpretation 7(1)
1.4.1 Hepatocyte suspension and plated hepatocyte system 7(1)
1.4.2 Physiologically based microfluidic systems 8(1)
1.5 Discussion and Commentary 8(5)
1.5.1 Hepatocyte suspension system 8(3)
1.5.2 Plated hepatocyte system 11(1)
1.5.3 Physiologically based microfluidic systems 12(1)
1.6 Summary 13(2)
References 15(4)
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(22)
2.1 Introduction 20(1)
2.2 Materials 21(1)
2.3 Methods 21(4)
2.3.1 Cultured cell system 21(3)
2.3.2 PAMPA system 24(1)
2.3.3 In vivo absorption measurements 25(1)
2.4 Data Acquisition, Anticipated Results, and Interpretation 25(5)
2.4.1 Data analysis 25(1)
2.4.2 Results and interpretation 26(4)
2.5 Discussion and Commentary 30(8)
2.5.1 Cell culture and PAMPA systems 30(5)
2.5.2 Absorption in experimental animals 35(1)
2.5.3 Rats 35(1)
2.5.4 Dogs 36(1)
2.5.5 Monkeys 37(1)
2.6 Summary 38(1)
References 38(3)
Chapter 3 Aggregating Brain Cell Cultures for Neurotoxicity Tests 41(20)
3.1 Introduction 42(1)
3.2 Experimental Design 43(1)
3.3 Materials 44(5)
3.3.1 Animals 44(1)
3.3.2 Special equipment 45(1)
3.3.3 Reagents 46(1)
3.3.4 Preparation of solutions and media 47(2)
3.4 Methods 49(6)
3.4.1 Washing and sterilizing the glassware 49(1)
3.4.2 Cell isolation and culture preparation 49(2)
3.4.3 Maintenance of aggregating brain cell cultures (media replenishment and subdivision) 51(1)
3.4.4 Preparation and treatment of replicate cultures 52(1)
3.4.5 Harvest of replicate cultures for various analytical procedures 53(1)
3.4.6 Examples of sample preparation and use for various analytical procedures 53(1)
3.4.7 Data Analysis 54(1)
3.5 Anticipated Results 55(2)
3.6 Discussion and Commentary 57(1)
3.7 Application Notes 57(1)
3.8 Summary Points 58(1)
Acknowledgments 59(1)
References 59(2)
Chapter 4 Approaches Towards a Multiscale Model of Systemic Inflammation in Humans 61(38)
4.1 Introduction 62(2)
4.2 Materials 64(1)
4.2.1 Human endotoxin model and data collection 64(1)
4.3 Methods 65(12)
4.3.1 Transcriptional dynamics and intrinsic responses 65(2)
4.3.2 Modeling inflammation at the cellular level 67(6)
4.3.3 Modeling inflammation at the systemic level 73(4)
4.4 Results 77(14)
4.4.1 Elements of the multiscale host response model of human inflammation 77(1)
4.4.2 Estimation of relevant model parameters 77(3)
4.4.3 Qualitative assessment of the model 80(11)
4.5 Conclusions 91(1)
Acknowledgments 91(1)
References 92(7)
Chapter 5 A Liposome Assay for Evaluating the Ocular Toxicity of Chemicals 99(16)
5.1 Introduction 100(1)
5.2 Experimental Design 101(1)
5.3 Materials 102(1)
5.4 Methods 103(5)
5.4.1 Preparation of calcein-loaded liposomes 103(1)
5.4.2 Separation of bulk calcein from loaded liposomes with Sephadex 104(2)
5.4.3 Ocular toxicity experiments using dye-loaded liposomes 106(2)
5.5 Data Acquisition, Anticipated Results, and Interpretation 108(1)
5.6 Discussion and Commentary 109(2)
5.7 Application Notes 111(1)
5.8 Summary Points 112(1)
References 113(2)
Chapter 6 Prediction of Potential Drug Myelotoxicity by In Vitro Assays on Hematopoietic Progenitors 115(18)
6.1 Introduction 116(1)
6.2 Experimental Design 116(1)
6.3 Materials 117(1)
6.3.1 Reagents 118(1)
6.4 Methods 118(6)
6.4.1 Preparation of methylcellulose stocks 118(1)
6.4.2 Source of murine hematopoietic progenitors 118(1)
6.4.3 Source of human hematopoietic progenitors 119(2)
6.4.4 Technical procedure for GM-CFU test 121(1)
6.4.5 Passing from screening phase to IC determination phase 122(1)
6.4.6 Incubator humidity test 122(1)
6.4.7 Scoring the colonies 123(1)
6.4.8 Criteria for colony counting 123(1)
6.5 Data Acquisition, Anticipated Results, and Interpretation 124(2)
6.5.1 Statistical guidelines 125(1)
6.6 Discussion and Commentary 126(2)
6.7 Application Notes 128(1)
6.8 Summary Points 128(2)
Acknowledgments 130(1)
References 130(3)
Chapter 7 Epigenetically Stabilized Primary Hepatocyte Cultures: A Potential Sensitive Screening Tool for Nongenotoxic Carcinogenicity 133(14)
7.1 Introduction 134(1)
7.2 Experimental Design 135(1)
7.3 Materials 135(4)
7.3.1 Reagents 135(4)
7.3.2 Facilities/Equipment 139(1)
7.4 Methods 139(2)
7.4.1 Isolation of hepatocytes from rat liver 139(2)
7.4.2 Cultivation of primary rat hepatocytes (Troubleshooting Table) 141(1)
7.5 Data Acquisition 141(1)
7.6 Anticipated Results and Interpretation 141(2)
7.7 Discussion and Commentary 143(1)
7.8 Application Notes 144(1)
7.9 Summary Points 144(1)
Acknowledgements 145(1)
References 145(2)
Chapter 8 A Statistical Method to Reduce In Vivo Product Testing Using Related In Vitro Tests and ROC Analysis 147(12)
8.1 Introduction 148(1)
8.2 Experimental Design 149(1)
8.3 Materials 149(1)
8.4 Methods 150(4)
8.4.1 Step-by-step protocol for the analysis of data using Analyse-It 152(2)
8.5 Results 154(1)
8.6 Discussion and Commentary 154(4)
8.6.1 Selecting the proper secondary test 154(1)
8.6.2 Determining the sample size for calibration and recalibration 155(1)
8.6.3 Regulatory concerns 156(1)
8.6.4 Determining the frequency of recalibration 156(1)
8.6.5 Determining the need for confirmatory testing 157(1)
8.6.6 Statistical analysis 157(1)
8.7 Summary Points 158(1)
Acknowledgments 158(1)
References 158(1)
Chapter 9 Application of the Benchmark Approach in the Correlation of In Vitro and In Vivo Data in Developmental Toxicity 159(12)
9.1 Introduction 160(2)
9.2 Materials and Methods 162(5)
9.2.1 Derivation of in vitro BMC and BMD values 163(1)
9.2.2 In vitro-in vivo correlation 164(3)
9.3 Discussion and Commentary 167(1)
References 168(3)
Chapter 10 Three-Dimensional Cell Culture of Canine Uterine Glands 171(12)
10.1 Introduction 172(1)
10.2 Materials 173(1)
10.2.1 Cell culture 173(1)
10.2.2 Histological preparation for light microscopy 173(1)
10.2.3 Histological preparation for electron microscopy 174(1)
10.3 Methods 174(4)
10.3.1 Cell culture 174(2)
10.3.2 Histological preparation for light microscopy 176(1)
10.3.3 Histological preparation for electron microscopy 177(1)
10.3.4 Imaging 178(1)
10.4 Anticipated Results 178(1)
10.5 Discussion and Commentary 178(2)
10.6 Application Notes 180(1)
10.7 Summary Points 181(1)
References 181(2)
Chapter 11 Markers for an In Vitro Skin Substitute 183(22)
11.1 Introduction 184(1)
11.2 Experimental Design 185(1)
11.3 Materials 185(3)
11.3.1 Human tissue-engineered skin substitute reconstructed by the self-assembly approach 185(3)
11.4 Methods 188(6)
11.4.1 Preparation of solutions and materials for the in vitro fabrication of human skin substitutes by the self-assembly approach 188(1)
11.4.2 In vitro fabrication of human skin substitutes by the self-assembly approach 189(2)
11.4.3 Tissue preservation and sectioning 191(1)
11.4.4 Preparation of solutions and materials for immunofluorescence 192(1)
11.4.5 Immunofluorescent labeling of human skin substitutes 192(1)
11.4.6 Histological analysis 193(1)
11.4.7 Transmission electron microscopy 193(1)
11.4.8 Statistical analysis 193(1)
11.5 Anticipated Results 194(4)
11.6 Discussion and Commentary 198(1)
11.7 Application Notes 199(1)
11.8 Summary Points 200(1)
Acknowledgments 201(1)
References 201(4)
Chapter 12 3D Culture of Primary Chondrocytes, Cartilage, and Bone/Cartilage Explants in Simulated Microgravity 205(22)
12.1 Introduction 206(2)
12.2 Experimental Design 208(3)
12.2.1 Culture models 208(1)
12.2.2 The RCCS bioreactor and its operational conditions 208(1)
12.2.3 Animals 209(2)
12.3 Materials 211(1)
12.3.1 Equipment for cell/tissue culture and preparation of samples 211(1)
12.3.2 Chemicals 211(1)
12.4 Methods 212(6)
12.4.1 Preparation of tissue explants 212(1)
12.4.2 Isolation of chondrocytes 212(2)
12.4.3 2D culture of isolated chondrocytes (traditional monolayer in static fluid conditions) 214(1)
12.4.4 3D culture of isolated chondrocytes (homotypic aggregates) 214(2)
12.4.5 3D culture of fragments of articular cartilage explants 216(1)
12.4.6 3D culture of undissected, complete proximal tibial epiphyses 217(1)
12.4.7 Histomorphological study of chondrocytes and cartilage tissue 217(1)
12.5 Anticipated Results 218(2)
12.6 Discussion 220(2)
12.6.1 Discussion of pitfalls 220(1)
12.6.2 General discussion and commentary 221(1)
12.7 Application Notes 222(1)
12.8 Summary Points 223(1)
Acknowledgments 224(1)
References 224(3)
Chapter 13 Alternatives for Absorption Testing 227(12)
13.1 Introduction 228(1)
13.2 Materials 229(1)
13.2.1 Franz diffusion cell 229(1)
13.2.2 Consumables 229(1)
13.2.3 Chemicals and solutions 229(1)
13.2.4 Technical equipment 230(1)
13.3 Methods 230(4)
13.3.1 Skin preparation 230(1)
13.3.2 Determination of skin penetration using the Franz cell setup 230(1)
13.3.3 Determination of skin permeation using the Franz cell setup 231(1)
13.3.4 Skin absorption studies with commercially available 3D skin models 231(1)
13.3.5 Quality control 231(1)
13.3.6 Data evaluation 232(1)
13.3.7 Biostatistics 233(1)
13.4 Results and Discussion 234(2)
13.5 Discussion of Pitfalls and Troubleshooting 236(1)
13.6 Summary 236(1)
References 237(2)
Chapter 14 A 3D Model of the Human Epithelial Airway Barrier 239(22)
14.1 Introduction 240(1)
14.2 Experimental Design 241(1)
14.3 Materials 241(2)
14.3.1 General materials 241(1)
14.3.2 Epithelial cell cultures鈥攖hawing 241(1)
14.3.3 Epithelial cell cultures鈥攃ulturing 241(1)
14.3.4 Isolation of monocyte-derived macrophages (MDM) and dendritic cells (MDDQ 242(1)
14.3.5 Triple cell coculture 242(1)
14.3.6 Transepithelial electrical resistance (TEER) measurements 242(1)
14.3.7 Staining for laser scanning microscopy (LSM) 243(1)
14.3.8 Embedding for transmission electron microscopy (TEM) 243(1)
14.4 Methods 243(10)
14.4.1 Epithelial cells 243(3)
14.4.2 Isolation of monocyte-derived macrophages (MDM) and dendritic cells (MDDC) 246(3)
14.4.3 TEER measurements 249(1)
14.4.4 Staining for LSM 250(1)
14.4.5 Fixation and embedding of cells for transmission electron microscopy (TEM) 251(2)
14.5 Anticipated Results 253(4)
14.6 Discussion and Commentary 257(1)
14.7 Application Notes 257(1)
14.8 Summary Points 257(1)
Acknowledgments 257(1)
References 258(3)
Chapter 15 Experimental Wear Assessment of Tibial Inserts for Total Knee Replacement 261(12)
15.1 Introduction 262(1)
15.2 Experimental Design 263(1)
15.3 Materials 263(2)
15.4 Methods 265(3)
15.4.1 Management of the specimens 265(1)
15.4.2 Wear test procedure 266(1)
15.4.3 Examination of worn tibial inserts surfaces 267(1)
15.5 Anticipated Results 268(1)
15.6 Discussion and Commentary 269(1)
15.7 Application Notes 269(1)
15.8 Summary Points 270(1)
Acknowledgments 271(1)
References 271(2)
About the Editors 273(2)
Index 275
- 名称
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