Isolated Hepatocytes: Preparation, Properties and Applications

Chapter 1. Background. 1.1. The development of the isolated hepatocyte preparation. 1.2. Factors involved in cellular organization and adhesion. 1.3. Mechanical methods for separation of parenchymal liver cells. 1.4. Development of enzymatic methods for isolated hepatocyte preparation. 1.5. Principles of cell separation. 1.6. Separation of cells from solid organs other than liver. 1.7. Advantages of the isolated hepatocyte preparation. 2. High-yield preparation of isolated hepatocytes from rat liver. 2.1 Introduction. 2.2. One-step method. 2.3. Two-step procedure. 2.4. Evaluation of common modifications to the recommended method. 2.5. Initial determination of cell quality. 2.6. Assessment of cell yield. 2.7. Reasons for unsatisfactory preparations. 2.8. Removal of damaged cells from the preparation. 2.9. Storage of hepatocytes. 3. Other methods for hypatocyte preparation. 3.1. Introduction. 3.2. Isolated hepatocyte preparation from liver slices. 3.3. Preparation of intact isolated hepatocytes without collagenase. 3.4. Preparation of isolated hepatocytes from species other than rat. 3.5. Preparation of hepatocytes from foetal or neonatal animals. 3.6. Preparation of hepatocytes from abnormal animals. 3.7. Preparation of suspensions of damaged hepatocytes. 3.8. Preparation and purification of non-parenchymal cells. 4. Assessment of integrity of isolated hepatocytes. 4.1. Introduction. 4.2. Measures of cellular integrity. 4.3. Additional methods for determining cellular integrity. 5. Microscopy of isolated hepatocytes. 5.1. Light microscopy of isolated hepatocytes. 5.2. Morphology of isolated hepatocytes by transmission electron microscopy. 5.3. Techniques. 5.4. Morphological studies yet to be exploited. 5.5. Summary of studies by transmission electron microscopy. 5.6. Scanning electron microscopy. 6. Biochemical properties. 6.1. Basis for expressing cell activity. 6.2. Measurement of cellular composition. 6.3. Cellular composition. 6.4. Experimental conditions. 6.5. Measurement of cellular O2-uptake. 6.6. Metabolic activities. 6.7. Transport activities. 7. Utilization of hepatocytes for drug studies. 7.1. Introduction. 7.2. Drug uptake and metabolism by isolated hepatocytes. 7.3. Isolated hepatocytes and drug toxicity. 8. Study the effects of hormones. 8.1. Optimal conditions for observing the effects of hormones. 8.3. Special conditions for the study of specific hormonal effects. 8.4. Action of insulin. 8.5. Action of steroid hormones. 8.6. Hormone effects on mitochondria isolated from hepatocytes. 9. Ca²⁺ ion transport and compartmentation. 9.1. Measurement of intracellular Ca²⁺. 9.2. Changes in Ca²⁺ movements monitored using ⁴⁵Ca²⁺. 9.3. Measurement of intracellular free Ca²⁺ using fluorescent indicators. 9.4. Determination of rates of Ca²⁺ inflow using glycogen phosphorylase or fluorescent Ca²⁺ indicators. 9.5. Assessment of available techniques for the measurement of Ca²⁺ in hepatocytes. 10. Hepatocyte isolation for primary culture and methods for non-adherent culture. 10.1. Introduction. 10.2. Alternatives to perfusion with collagenase in isolating cells for culture. 10.3. Choice of collagenase perfusion isolation procedure. 10.4. Preserving sterility of cell preparations. 10.5. A procedure for hepatocyte isolation. 10.6. Suspension culture of hepatocytes. 10.7. Other non-adherent culture methods. 11. Monolayer culture of hepatocytes. 11.1 Characteristics of adult primary monolayer cultures: an overview. 11.2. Attachtment of hepatocytes to substrata for monolayer culture. 11.3. Preparation, use and functional effects of specific substrata. 11.4. Choice of Culture Media. 11.5. Non-physiological supplements to culture media. 11.6. Homologous cell interactions. 11.7. Heterologous cell interactions. 11.8. Selecting appropriate methods for monolayer culture. 11.9. Variations on monolayer culture. 11.10. Cultures of hepatocytes from foetal, neonatal and suckling rats. 11.11. Culture of hepatocytes from other species. 11.12. Some commonly used experimental methods in studies with hepatocyte monolayers. 12. Selected specialized techniques. 12.1. Introduction. 12.2. Sub-cellular fractionation of hepatocytes. 12.3. Methods of subcellular fractionation. 12.4. Homogenization of isolated liver cell suspensions. 12.5. Preparation of cell fragments ('cytospheres'). 12.6. Preparation of plasma membrane fractions. 12.7. Permeabilized Cells. 12.8. Perifusion of hepatocytes. 12.9. Measurement of cellular membrane potentials. 12.10. Measurement of intracellular pH. 12.11. Tramsportation of hepatocytes. 12.12. Studies with hepatocyte 'doublets'. 12.13. Separation of periportal and perivenous hepatocytes. 12.14. Future developments. Appendix 1. Composition of media. Appendix 2. Addresses of suppliers and manufacturers. References. Subject Index.