Live Cell Assays

Christophe Furger is a cell biologist, Doctor of Pierre et Marie Curie University (Sorbonne Universities, Paris, France) and Director of Research and Development at L.E.D. He has been working since 1995 on the conception of cell assays. He currently heads a public/private research program in toxicity at the LAAS/CNRS lab (Toulouse, France).

• Foreword xiiiAcknowledgments xixAbbreviations xxiList of Cell Assays xxiiiIntroduction xxviiChapter 1. Principles and Position 11.1. Live cell assay principles 11.2. Application areas 31.3. Positioning 51.3.1. Definition and typology of cell tests 61.3.2. The regulatory and industrial dimension 81.4. Market 91.5. Competitive advantages 121.5.1. Cells are live information models 121.5.2. Development: high throughput 131.5.3. Development: multiplex analysis 131.5.4. Development: miniaturization 141.5.5. Development: molecular engineering 141.5.6. Development: standardization 141.6. Can measurements of cells in culture be extrapolated to effects in the organism? 151.6.1. Toxicokinetics 151.6.2. Components of the immune system 161.6.3. Biotransformation 161.6.4. The macrocellular environment 161.7. Limits 171.7.1. Importance of cellular microenvironment 171.7.2. Other limits 19Chapter 2. History and State of the Art 212.1. Origins of cell culture 212.1.1. Pioneering studies 222.1.2. Alexis Carrel 232.1.3. Were Dr Carrel’s cells immortal? 252.2. The HeLa line and the first applications of cell culture 272.2.1. A vaccine against poliomyelitis 292.2.2. Cells in space 292.2.3. Cell cloning 302.3. New cell lines 302.3.1. The CHO line 302.3.2. An increasing number of cell lines 312.4. Cross-contamination 322.5. Cell lines, an ethical issue 352.6. The first generation of cell assays (1969–1983) 372.6.1. The karyotype test 382.6.2. The MTT assay 392.6.3. The NRU test 412.7. The first target of regulatory assays: genotoxicity (1983–1986) 422.7.1. Ames test (OECD guideline 471) 432.7.2. In vitro mammalian chromosome aberration test (OECD guideline 473) 442.7.3. In vitro mammalian cell gene mutation test (OECD guideline 476) 452.7.4. In vitro sister chromatid exchange assay in mammalian cells (OECD guideline no. 479) 462.7.5. DNA damage and repair, unscheduled DNA synthesis in mammalian cells (OECD guideline 482) 47Chapter 3. Cell Models and Technologies 493.1. Fluorescence and bioluminescence 503.1.1. Green fluorescent protein 513.1.2. BRET 533.1.3. FRET 553.1.4. Other applications of GFP 573.1.5. The reporter gene approach 583.2. Impedance variation in cell population 603.3. Optical signals modified by state of cells 623.4. Cellular autofluorescence 653.4.1. The case of chlorophyll 663.5. The different cell models and culture modes available 673.5.1. Immortalized lines 683.5.2. Primary cells 693.5.3. Three-dimensional cell culture 69Chapter 4. Loss of Cell Homeostasis: Applications in Toxicity Measurement 714.1. What relevant information to use in the living cell? 714.2. Lysosomal activity 734.3. Redox balance and oxidative stress 764.4. Integrity of the plasma membrane 804.5. Cellular efflux 844.6. Homeostasis of ion exchanges 894.6.1. The calcium ion 894.6.2. Maintenance of membrane potential 914.7. Metabolism and cell respiratory activity 924.8. Genotoxicity 954.9. Apoptosis 97Chapter 5. The Replacement of Animal Testing: A Driving Force in Live Cell Assay Development 1035.1. On the pertinence of in vitro assays 1045.2. On the pertinence of animal tests 1055.3. The problem with extrapolation 1065.3.1. The interspecies barrier 1065.3.2. The striking example of TGN1412 1075.4. Toxicological assessment of substances 1095.5. Irritation and eye corrosion: the long (ongoing) quest for an alternative to the Draize test 1115.5.1. The CM test 1125.5.2. Ex vivo approaches 1135.5.3. 3D culture models 1145.5.4. Recent attempts and validations 1155.6. Measurement alternatives for skin absorption, corrosion and irritation (2004–2010) 1165.6.1. Skin absorption: in vitro method (OECD guideline no. 428) 1175.6.2. Reconstituted skin models for corrosion and irritation 1175.6.3. In vitro skin corrosion: human skin model test (OECD guideline no. 431) 1185.6.4. In vitro membrane barrier test method for skin corrosion (OECD guideline 435) 1215.6.5. In vitro skin irritation: reconstructed human epidermis test method (OECD guideline no. 439) 1215.7. The live cell test for phototoxicity measurement (2004) 1225.8. Assays for endocrine disruptor tracking (2009–2011) 1235.8.1. Detection of estrogenic agonist-activity of chemicals (OECD guideline 455) 1245.8.2. H295R steroidogenesis assay (OECD guideline 456) 1245.9. The four last live cell assays to be validated (2012–2015) 1255.9.1. Eye corrosion: fluorescein leakage test method (OECD guideline 460) 1255.9.2. Mammalian cell micronucleus test (OECD guideline 487) 1265.9.3. ARE-Nrf2 luciferase test method for in vitro skin sensitization (OECD guideline no 442D) 1275.9.4. Short-time exposure in vitro test method for identifying (1) chemicals inducing serious eye damage and (2) chemicals not requiring classification for eye irritation or serious eye damage (OECD guideline 491) 127Chapter 6. Regulatory Applications and Validation 1296.1. Brief history of the validation process in Europe 1296.2. The validation process of a live cell assay 1306.3. Live cell assays adopted by the OECD 1326.4. The future of regulatory cell tests: the TOX21 and SEURAT programs 1346.4.1. TOX21, a new paradigm in the assessment of health and environmental risks 1346.4.2. The SEURAT-1 program (2011–2016) 1386.5. The REACH regulatory context 139 6.5.1. Assessment approach by weight of evidence (WoE) 1406.5.2. Up-date on the use of live cell assays under REACH 1406.5.3. Acute toxicity 1416.5.4. Skin corrosion and irritation 1426.5.5. Eye irritation and severe damage 1426.5.6. Skin sensitization 1426.5.7. Repeated doses (long-term effects) 1426.5.8. Genotoxicity 1436.5.9. Reproductive toxicity (reprotoxicity) 1436.5.10. Carcinogenicity 1436.5.11. Bioaccumulation and toxicity in fish 1446.5.12. Long-term toxicity and reprotoxicity in birds 1446.6. Implementation of the 7th amendment to the Cosmetics Directive 1446.6.1. Acute toxicity 1456.6.2. Eye corrosion and irritation 1456.6.3. Skin irritation and corrosion 1466.6.4. Skin sensitization 1466.6.5. Genotoxicity 1476.6.6. Skin absorption 1476.7. Food safety and biocides directive 1476.7.1. Food safety 1476.7.2. The biocides directive 148Chapter 7. Cell Signaling: At the Heart of Functional Assays for Industrial Purposes 1497.1. Membrane receptors, the primary target of drugs 1497.1.1. Development of the therapeutic target/receptor concept 1507.1.2. Purification, sequencing and heterologous expression 1517.1.3. The therapeutic importance of seven transmembrane domain receptors 1527.2. Second messenger, base unit of the functional live cell assay 1537.2.1. The second messenger concept 1537.2.2. Adenylyl cyclase and phosphodiesterase regulate the concentration of cyclic AMP 1557.3. The concept of cell transduction 1567.3.1. The protein kinase A, the (near) universal target of cyclic AMP 1577.3.2. Decrypting the transduction pathways 1587.3.4. G proteins, the missing link in cell transduction 1607.3.5. Connection between transduction and genic expression 1617.4. The transduction pathways used in the context of live cell assays 1627.4.1. First level of regulation – activation of the transduction pathway 1637.4.2. Second level of regulation – desensitization and recycling 1647.4.3. Third level of regulation – allosteric modulation 165Chapter 8. Applications in New Drug Discovery 1678.1. High-throughput screening, the leading market sector for cell assays 1678.1.1. The role of cell assays in screening programs 1698.1.2. The contribution of functional cell assays 1718.1.3. Exploitation of transduction pathways 1718.2. Measurements in the immediate environment of receptors 1738.2.1. Assays on receptors 1738.2.2. β-arrestin activity assays 1748.3. Measuring cyclic AMP 1778.3.1. Classic cyclic AMP assays on cellular lysates 1778.3.2. Cyclic AMP assays on live culture cells 1808.4. Measurement of the PKC pathway and discrimination of the PKA/PKC pathways 1838.4.1. IP3 measurement tests 1838.4.2. Assays for the measurement of Ca2+ 1838.4.3. Discrimination between the cyclic AMP and IP3/Ca2+ pathways by label-free methods 1848.5. Measurement of distal signals  1858.6. Cell assays concerning other therapeutic targets 1868.6.1. Measurement on ion channels 1868.6.2. Measurements on receptor tyrosine kinases (RTK) 1888.7. Pharmacokinetics (ADME) in vitro 1918.7.1. M for metabolism 1928.7.2. A for absorption 1938.7.3. T for toxicity 195Chapter 9. Impact on Health and the Environment 1979.1. Patient diagnosis 1979.1.1. Cytogenetics 1989.1.2. Diagnosis of tuberculosis 2009.1.3. Cell assay for the detection of pyrogenic substances 2019.1.4. Cell assays for predicting efficacy of chemotherapy 2039.2. Military programs 2049.2.1. Detection and screening of botulinum toxin inhibitors 2059.2.2. Antibody-based toxin neutralization assays (TNA): application on anthrax and ricin 2089.2.3. Field measurement of water potability 2099.3. Pollution and quality of environment 2119.3.1. The MicroTox assay 2119.3.2. Mobility of the Daphnia test 2129.3.3. Fish embryo acute toxicity (FET) test (OECD guideline no. 236) 2139.3.4. The DR CALUX assay 2149.3.5. Biomonitoring and field issues 215Chapter 10. Outlook 21910.1. Stem cells, an opportunity for the future of cell assays 21910.2. Organs-on-a-chip 22210.2.1. Homo chippiens 22410.2.2. The contribution of PBPK models 22510.3. Conclusion 226Bibliography 229Index 247