Cover

Title Page

Copyright

Contents

Foreword

A researcher’s view

A consultant’s view

The author

Acknowledgments

Abbreviations

List of Cell Assays

Introduction

1: Principles and Position

1.1. Live cell assay principles

1.2. Application areas

1.3. Positioning

1.3.1. Definition and typology of cell tests

1.3.2. The regulatory and industrial dimension

1.4. Market

1.5. Competitive advantages

1.5.1. Cells are live information models

1.5.2. Development: high throughput

1.5.3. Development: multiplex analysis

1.5.4. Development: miniaturization

1.5.5. Development: molecular engineering

1.5.6. Development: standardization

1.6. Can measurements of cells in culture be extrapolated to effects in the organism?

1.6.1. Toxicokinetics

1.6.2. Components of the immune system

1.6.3. Biotransformation

1.6.4. The macrocellular environment

1.7. Limits

1.7.1. Importance of cellular microenvironment

1.7.2. Other limits

2: History and State of the Art

2.1. Origins of cell culture

2.1.1. Pioneering studies

2.1.2. Alexis Carrel

2.1.3. Were Dr Carrel’s cells immortal?

2.2. The HeLa line and the first applications of cell culture

2.2.1. A vaccine against poliomyelitis

2.2.2. Cells in space

2.2.3. Cell cloning

2.3. New cell lines

2.3.1. The CHO line

2.3.2. An increasing number of cell lines

2.4. Cross-contamination

2.5. Cell lines, an ethical issue

2.6. The first generation of cell assays (1969–1983)

2.6.1. The karyotype test

2.6.2. The MTT assay

2.6.3. The NRU test

2.7. The first target of regulatory assays: genotoxicity (1983–1986)

2.7.1. Ames test (OECD guideline 471)

2.7.2. In vitro mammalian chromosome aberration test (OECD guideline 473)

2.7.3. In vitro mammalian cell gene mutation test (OECD guideline 476)

2.7.4. In vitro sister chromatid exchange assay in mammalian cells (OECD guideline no. 479)

2.7.5. DNA damage and repair, unscheduled DNA synthesis in mammalian cells (OECD guideline 482)

3: Cell Models and Technologies

3.1. Fluorescence and bioluminescence

3.1.1. Green fluorescent protein

3.1.2. BRET

3.1.3. FRET

3.1.4. Other applications of GFP

3.1.5. The reporter gene approach

3.2. Impedance variation in cell population

3.3. Optical signals modified by state of cells

3.4. Cellular autofluorescence

3.4.1. The case of chlorophyll

3.5. The different cell models and culture modes available

3.5.1. Immortalized lines

3.5.2. Primary cells

3.5.3. Three-dimensional cell culture

4: Loss of Cell Homeostasis: Applications in Toxicity Measurement

4.1. What relevant information to use in the living cell?

4.2. Lysosomal activity

4.3. Redox balance and oxidative stress

4.4. Integrity of the plasma membrane

4.5. Cellular efflux

4.6. Homeostasis of ion exchanges

4.6.1. The calcium ion

4.6.2. Maintenance of membrane potential

4.7. Metabolism and cell respiratory activity

4.8. Genotoxicity

4.9. Apoptosis

5: The Replacement of Animal Testing: A Driving Force in Live Cell Assay Development

5.1. On the pertinence of in vitro assays

5.2. On the pertinence of animal tests

5.3. The problem with extrapolation

5.3.1. The interspecies barrier

5.3.2. The striking example of TGN1412

5.4. Toxicological assessment of substances

5.5. Irritation and eye corrosion: the long (ongoing) quest for an alternative to the Draize test

5.5.1. The CM test

5.5.2. Ex vivo approaches

5.5.3. 3D culture models

5.5.4. Recent attempts and validations

5.6. Measurement alternatives for skin absorption, corrosion and irritation (2004–2010)

5.6.1. Skin absorption: in vitro method (OECD guideline no. 428)

5.6.2. Reconstituted skin models for corrosion and irritation

5.6.3. In vitro skin corrosion: human skin model test (OECD guideline no. 431)

5.6.4. In vitro membrane barrier test method for skin corrosion (OECD guideline 435)

5.6.5. In vitro skin irritation: reconstructed human epidermis test method (OECD guideline no. 439)

5.7. The live cell test for phototoxicity measurement (2004)

5.8. Assays for endocrine disruptor tracking (2009–2011)

5.8.1. Detection of estrogenic agonist-activity of chemicals (OECD guideline 455)

5.8.2. H295R steroidogenesis assay (OECD guideline 456)

5.9. The four last live cell assays to be validated (2012–2015)

5.9.1. Eye corrosion: fluorescein leakage test method (OECD guideline 460)

5.9.2. Mammalian cell micronucleus test (OECD guideline 487)

5.9.3. ARE-Nrf2 luciferase test method for in vitro skin sensitization (OECD guideline no 442D)

5.9.4. 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)

6: Regulatory Applications and Validation

6.1. Brief history of the validation process in Europe

6.2. The validation process of a live cell assay

6.3. Live cell assays adopted by the OECD

6.4. The future of regulatory cell tests: the TOX21 and SEURAT programs

6.4.1. TOX21, a new paradigm in the assessment of health and environmental risks

6.4.2. The SEURAT-1 program (2011–2016)

6.5. The REACH regulatory context

6.5.1. Assessment approach by weight of evidence (WoE)

6.5.2. Up-date on the use of live cell assays under REACH

6.5.3. Acute toxicity

6.5.4. Skin corrosion and irritation

6.5.5. Eye irritation and severe damage

6.5.6. Skin sensitization

6.5.7. Repeated doses (long-term effects)

6.5.8. Genotoxicity

6.5.9. Reproductive toxicity (reprotoxicity)

6.5.10. Carcinogenicity

6.5.11. Bioaccumulation and toxicity in fish

6.5.12. Long-term toxicity and reprotoxicity in birds

6.6. Implementation of the 7th amendment to the Cosmetics Directive

6.6.1. Acute toxicity

6.6.2. Eye corrosion and irritation

6.6.3. Skin irritation and corrosion

6.6.4. Skin sensitization

6.6.5. Genotoxicity

6.6.6. Skin absorption

6.7. Food safety and biocides directive

6.7.1. Food safety

6.7.2. The biocides directive

7: Cell Signaling: At the Heart of Functional Assays for Industrial Purposes

7.1. Membrane receptors, the primary target of drugs

7.1.1. Development of the therapeutic target/receptor concept

7.1.2. Purification, sequencing and heterologous expression

7.1.3. The therapeutic importance of seven transmembrane domain receptors

7.2. Second messenger, base unit of the functional live cell assay

7.2.1. The second messenger concept

7.2.2. Adenylyl cyclase and phosphodiesterase regulate the concentration of cyclic AMP

7.3. The concept of cell transduction

7.3.1. The protein kinase A, the (near) universal target of cyclic AMP

7.3.2. Decrypting the transduction pathways

7.3.4. G proteins, the missing link in cell transduction

7.3.5. Connection between transduction and genic expression

7.4. The transduction pathways used in the context of live cell assays

7.4.1. First level of regulation – activation of the transduction pathway

7.4.2. Second level of regulation – desensitization and recycling

7.4.3. Third level of regulation – allosteric modulation

8: Applications in New Drug Discovery

8.1. High-throughput screening, the leading market sector for cell assays

8.1.1. The role of cell assays in screening programs

8.1.2. The contribution of functional cell assays

8.1.3. Exploitation of transduction pathways

8.2. Measurements in the immediate environment of receptors

8.2.1. Assays on receptors

8.2.2. β-arrestin activity assays

8.3. Measuring cyclic AMP

8.3.1. Classic cyclic AMP assays on cellular lysates

8.3.2. Cyclic AMP assays on live culture cells

8.4. Measurement of the PKC pathway and discrimination of the PKA/PKC pathways

8.4.1. IP3 measurement tests

8.4.2. Assays for the measurement of Ca2+

8.4.3. Discrimination between the cyclic AMP and IP3/Ca2+ pathways by label-free methods

8.5. Measurement of distal signals

8.6. Cell assays concerning other therapeutic targets

8.6.1. Measurement on ion channels

8.6.2. Measurements on receptor tyrosine kinases (RTK)

8.7. Pharmacokinetics (ADME) in vitro

8.7.1. M for metabolism

8.7.2. A for absorption

8.7.3. T for toxicity

9: Impact on Health and the Environment

9.1. Patient diagnosis

9.1.1. Cytogenetics

9.1.2. Diagnosis of tuberculosis

9.1.3. Cell assay for the detection of pyrogenic substances

9.1.4. Cell assays for predicting efficacy of chemotherapy

9.2. Military programs

9.2.1. Detection and screening of botulinum toxin inhibitors

9.2.2. Antibody-based toxin neutralization assays (TNA): application on anthrax and ricin

9.2.3. Field measurement of water potability

9.3. Pollution and quality of environment

9.3.1. The MicroTox assay

9.3.2. Mobility of the Daphnia test

9.3.3. Fish embryo acute toxicity (FET) test (OECD guideline no. 236)

9.3.4. The DR CALUX assay

9.3.5. Biomonitoring and field issues

10: Outlook

10.1. Stem cells, an opportunity for the future of cell assays

10.2. Organs-on-a-chip

10.2.1. Homo chippiens

10.2.2. The contribution of PBPK models

10.3. Conclusion

Bibliography

Index

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