Isolated Hepatocytes: Preparation, Properties and Applications (eBook)

Preparation, Properties and Applications

G.J. Barritt, M.N. Berry, A.M. Edwards (Autoren)

R. H. Burdon (Herausgeber)

eBook Download: PDF

1991 | 1. Auflage
459 Seiten
Elsevier Science (Verlag)
978-0-08-085890-6 (ISBN)

Infos

This book sets out clearly and effectively the preparation and working methods of laboratory techniques involving isolated hepatocytes and will make life easier for every laboratory worker concerned with these techniques.

Inhalt

Front Cover 1
Isolated Hepatocytes Preparation, Properties and Applications 4
Copyright Page 5
Contents 12
Preface 6
Acknowledgements 10
Abbreviations 22
Chapter 1. Background 24
1.1. The development of the isolated hepatocyte preparation 24
1.2. Factors involved in cellular organization and adhesion 25
1.3. Mechanical methods for separation of parenchymal liver cells 27
1.4. Development of enzymatic methods for isolated hepatocyte preparation 29
1.5. Principles of cell separation 32
1.6. Separation of cells from solid organs other than liver 35
1.7. Advantages of the isolated hepatocyte preparation 37
Chapter 2. High-yield preparation of isolated hepatocytes from rat liver 38
2.1. Introduction 38
2.2. One-step method 39
2.3. Two-step procedure 55
2.4. Evaluation of common modifications to the recommended method 59
2.5. Initial determination of cell quality 68
2.6. Assessment of cell yield 71
2.7. Reasons for unsatisfactory preparations 76
2.8. Removal of damaged cells from the preparation 78
2.9. Storage of hepatocytes 80
Chapter 3. Other methods for hepatocyte isolation 82
3.1. Introduction 82
3.2. Preparation of isolated hepatocytes from liver slices 82
3.3. Preparation of intact isolated hepatocytes without collagenase 84
3.4. Preparation of isolated hepatocytes from species other than rat 87
3.5. Preparation of hepatocytes from foetal or neonatal animals 97
3.6. Preparation of hepatocytes from abnormal animals 99
3.7. Preparation of suspensions of damaged hepatocytes 100
3.8. Preparation and purification of non-parenchymal cells 103
Chapter 4. Assessment of integrity of isolated hepatocytes 106
4.1. Introduction 106
4.2. Measures of cellular integrity 108
4.3. Additional methods for determining cellular integrity 117
4.4. Assesment of cellular integrity following cell incubations 120
Chapter 5. Microscopy of isolated hepatocytes 122
5.1. Light microscopy of isolated hepatocytes 122
5.2. Morphology of isolated hepatocytes by transmission electron microscopy 126
5.3. Techniques 135
5.4. Morphological studies yet to be exploited 138
5.5. Summary of studies by transmission electron microscopy 140
5.6. Scanning electron microscopy 140
Chapter 6. Biochemical properties 144
6.1. Basis for expressing cell activity 144
6.2. Measurement of cellular composition 149
6.3. Cellular composition 163
6.4. Experimental conditions 171
6.5. Measurement of cellular O2-uptake 183
6.6. Metabolic activities 191
6.7. Transport activities 199
6.8. Analysis of radiolabelled compounds 201
Chapter 7. Utilization of hepatocytes for drug studies 202
7.1. Introduction 202
7.2. Drug uptake and metabolism by isolated hepatocytes 205
7.3. Isolated hepatocytes and drug toxicity 211
7.4 Metabolism and effects of ethanol 222
Chapter 8. Study of the effects of hormones 224
8.1. Hormonal responses exhibited by isolated hepatocytes 224
8.2. Optimal conditions for observing the effects of hormones 225
8.3. Special conditions for the study of specific hormonal effects 231
8.4. Actions of insulin 232
8.5. Action of steroid hormones 233
8.6. Hormone effects on mitochondria isolated from hepatocytes 234
Chapter 9. Ca2+ ion transport and compartmentation 238
9.1. Measurement of intracellular Ca2+ 238
9.2. Changes in Ca2+ movement monitored using 45Ca2+ 242
9.3. Measurement of intracellular free Ca2+ using fluorescent indicators 247
9.4. Determination of rates of Ca2+ inflow using glycogen phosphorylase or fluorescent Ca2+ indicators 252
9.5. Assessment of available techniques for the measurement of Ca2+ in hepatocytes 258
Chapter 10. Hepatocyte isolation for primary culture and methods for non-adherent culture 260
10.1. Introduction 260
10.2. Alternatives to perfusion with collagenase in isolating cells for culture 262
10.3. Choice of collagenase perfusion isolation procedure 264
10.4. Preserving sterility of cell preparations 269
10.5. A procedure for hepatocyte isolation 271
10.6. Suspension culture of hepatocytes 275
10.7. Other non-adherent culture methods 283
Chapter 11. Monolayer culture of heputocytes 288
11.1. Characteristics of adult primary monolayer cultures: an overview 288
11.2. Attachment of hepatocytes to substrata for monolayer culture 301
11.3. Preparation, use and functional effects of specific substrata 305
11.4. Choice of culture media 315
11.5. Non-physiological supplements to culture media 338
11.6. Homologous cell interactions 342
11.7. Heterologous cell interactions 344
11.8. Selecting appropriate methods for monolayer culture 349
11.9. Variations on monolayer culture 356
11.10. Culture of hepatocytes from foetal, neonatal and suckling rats 360
11.11. Culture of hepatocytes from other species 365
11.12. Some commonly used experimental methods in studies with hepatocyte monolayers 370
Chapter 12. Selected specialized techniques 378
12.1. Introduction 378
12.2. Sub-cellular fractionation of hepatocytes 378
12.3. Methods of subcellular fractionation 380
12.4. Homogenization of isolated liver cell suspensions 387
12.5. Preparation of cell fragments ('cytospheres') 388
12.6. Preparation of plasma membrane fractions 395
12.7. Permeabilized cells 395
12.8. Perifusion of hepatocytes 401
12.9. Measurement of cellular membrane potentials 407
12.10. Measurement of intracellular pH 413
12.11. Transplantation of hepatocytes 414
12.12. Studies with hepatocyte 'doublets' 418
12.13. Separation of periportal and perivenous hepatocytes 420
12.14. Future developments 422
Appendix 1 Composition of media 424
Appendix 2 Addresses of suppliers and manufacturers 426
References 432
Subject Index 464

Produkt-Details

• Erscheint lt. Verlag: 30.9.1991
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-08-085890-2 / 0080858902
• ISBN-13: 978-0-08-085890-6 / 9780080858906