Live Cell Assays

From Research to Regulatory Applications

Christophe Furger

• Publisher: Wiley-ISTE
• Year: 2016
• ISBN:
  9781119330172
• Edition: 1
• Topic: Sciences biologiques
• Subtopic: Biologie cellulaire

Cell assays include all methods of measurements on living cells. Confined for a long time to research laboratories, these emerging methods have, in recent years, found industrial applications that are increasingly varied and, from now on, regulatory.

Based on the recent explosion of knowledge in cell biology, the measurement of living cells represents a new class of industry-oriented research tests, the applications of which continue to multiply (pharmaceuticals, cosmetics, environment, etc.). Cellular tests are now being positioned as new tools at the interface between chemical methods, which are often obsolete and not very informative, and methods using animal models, which are expensive, do not fit with human data and are widely discussed from an ethical perspective. Finally, the development of cell assays is currently being strengthened by their being put into regulatory application, particularly in Europe through the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) and cosmetic directives.

1
Principles and Position

1.1. Live cell assay principles

Cell culture aims to isolate cells from organisms then to keep them alive for experimental uses. Cell models vary widely. For practical reasons, the available human cells are, for the most part, tumorous in origin, having been immortalized so as to remain living for numerous generations. Culture cells can also be natural, which means that cells are collected in tissue or in organs for the purposes of an experiment. These are known as primary cells. Additionally, cells can be modified by a bioengineer so as to express genes that they did not originally possess. These are known as transgenic models.

In any case, cells must adapt to their new way of existing in vitro, a world in which they can no longer benefit from the multiple opportunities of complex exchange and communication inherent to their natural environment. Consequently, their behavior in culture is typically remote from the role they fulfilled as part of the organism.

Cell culture has been understood for over half a century. This long history provides it with a backlog of numerous applications spanning more than just cell assays (Figure 1.1).

Historically, cell cultures have acted as models for fundamental research and knowledge acquisition, particularly in cellular and molecular biology. Transgenic cultures, primarily based on the Chinese Hamster Ovary model (see section 2.3), have since been used as “factories” for the mass production of biopharmaceutics such as hormones or antibodies. More recently, cells have been in the limelight due to the first developments in cell therapy, a sector with great potential though very much still in development.

Figure 1.1. Position of cell assays within the various application areas of cell culture

And finally, cell cultures have been used to perform evaluations and measurements. This is the area of cell assays.

The principle of cell assays is founded on the evaluation of an experimental condition, a cell model and a means of measurement. The choice of cell model is essential. Unlike other industrial or clinical applications, cell assays use the cell only to produce information. Accordingly, the cell model is chosen for its faithful representation of the biological context in which the information is being sought. The matching of the cell model to the experimental objective is clearly the key to evaluating if a proposed cell assay is fit for purpose. Any discussion about measurement quality will be dependent on the demonstration of this match.

This difficulty can be eased by considering the cell as representing a certain level of information to be reached. For example, the information in the living model is capable of integrating the effect of the experimental condition in the form of a global response. This is often the case in studies of cytotoxicity where the signals of interest are limited to global effects such as proliferation, apoptosis, alteration in DNA or membrane integrity. In such cases, the choice of the model ultimately counts for little. The response measured is shared by the vast majority of cell types. Ultimately, numerous assays work in this way, utilizing the living cell by default, as a simple demonstration of the effect sought on a living model.

However, some specific properties can be used for application purposes. These properties are dependent on the level of differentiation that the cell managed to retain in culture. These levels increase the pertinence of the cell assay’s information level. For example, neurons or cardiac cells can be used to measure signals of electrical excitability, liver cells can be used to metabolize and thereby activate or deactivate a compound’s toxicity.

To study the expression of a specific signal typically requires genome modification by transgenesis, which is the preferred method of orientating a cell toward a particular phenotype. Cell models developed in this way will have acquired a truly specific response. This strategy is widely employed in the pharmaceutical industry to create models that coexpress the therapeutic target of interest and the measured signal, based for the most part on fluorescent or luminescent proteins.

Notwithstanding, the question of the measurement method is more readily resolved. These methods are numerous and benefitted greatly from advances in molecular biology through the decade 1985–1995. Over the last 20 years, these advances have been consolidated while providing demonstrations of their viability.

1.2. Application areas

Live cell assays can be broadly categorized according to three areas of application (Table 1.1):

1. – cytotoxicity measurement;
2. – discovery of new medicines;
3. – diagnostics (pathological, military and environmental).

Cytotoxicity measurement represents a driving force in the development of live cell assays. Indeed, in a certain way, this is their natural application. There are two reasons for this: measuring cytotoxicity is above all a major issue in public health and increasingly so due to the modern preoccupation for pollution. However, cytotoxicity is difficult to evaluate without engaging living models as toxicity must be expressed. Then the cell becomes an essential target for toxicity. In the first instance, this typically manifests by a loss of homeostasis (reactive oxygen species generation, increase in ATP consumption, loss of membrane integrity, mitochondrial changes, DNA changes). The living cell in culture has proved itself to be an attractive model for such assessments. Homeostasis measurement methods are both reliable and numerous. Today, they cover the entirety of intimate, inner cell functions (see Chapter 4). Furthermore the cell is rendered fragile by being maintained in culture, often presenting high susceptibility to the effect of exogenous compounds.

Live cell tests are widely employed at various stages in the discovery of new medicines, from identifying therapeutic targets to validating compounds of interest. The essential area of application, in volume at least, is molecular screening. The strategy here consists of creating a cell model expressing the therapeutic target, and then employing it to select compounds of interest from chemical libraries according to both their capacity to bind themselves to the target in question and obtaining the expected response. Screening can be at high or ultrahigh throughput (with libraries of several thousands or hundreds of thousands of compounds) or high content (multiplex analysis of different cell parameters by image analysis). This vast area of application will be treated in more depth in Chapter 8.

Diagnostics represent the third main area of application for cell assays. The three main subsets of this area are public health, military programs and the environment. In public health, diagnostics consist of putting cells into cultures that have only recently been extracted from patients (see section 9.1). The signals observed will typically be genomic (karyotype), infectious (presence of antibodies) or therapeutic (efficacy in chemotherapy). Applications in diagnostics have a long history, with the first assays (see section 2.2) being perfected in the 1950s within the context of programs studying poliomyelitis. Military programs use assays to protect soldiers’ health in the theater of operations (see section 9.2). The principle is to ensure the extemporaneous identification of toxins in the event of bioterrorist acts. The environmental issue joins the military one but on a far more vast panel of polluting compounds (see section 9.3). The measuring technologies employed here are the same as other applications, albeit with cell models approaching those used in ecology (fish, bacteria, algae, etc.).

Table 1.1. Main applications of live cell assays

Cytotoxicity measurement	Regulatory (health checks on chemical or cosmetic products)
	Evaluation of drug candidates (pharmaceutic industry)
Diagnostics	Pathological
	Military (bioterrorism, theater of operations)
	Environmental (pollution)
Drug Discovery	High content or throughput screening
	Pharmacokinetics (ADME)

1.3. Positioning

Cell assays are positioned at the half-way point between physicochemical tests, which measure the presence of substances or specific activities in abiotic systems, and animal tests, which are of a functional nature and provide answers at the organism level. Indeed, both of these varieties of tests are historically well-established. In the current industrial and regulatory landscape, cell tests are still considered as something of an alternative strategy with both advantages and disadvantages.

Physicochemical tests are mono-informative and quantitative by their very nature. While they measure the presence of molecular species in a clear, precise and standardizable way, they do not supply any indication on the effect or impact of this presence on the living being. Furthermore, they are often bonded to specific molecular species. By and large, they find only what they look for. Ultimately, these tests give rise to throughput problems and often require support from more onerous and expensive technologies.

On the other hand, animal tests are qualitative. The main interest of these tests is their capacity for evaluating the effect or the impact of a chemical species or mixture on an organism. With regards to effects on humankind, the extrapolation of these tests is dubious. Furthermore, they are very poorly adapted to high throughput, very hard to standardize and extremely expensive. They also give rise to major ethical problems that will be addressed in depth later.

The final goal of live cell assays is to sur...

Table of contents

• Cover
• Table of Contents
• Title
• Copyright
• Foreword
• Acknowledgments
• Abbreviations
• List of Cell Assays
• Introduction
• 1 Principles and Position
• 2 History and State of the Art
• 3 Cell Models and Technologies
• 4 Loss of Cell Homeostasis: Applications in Toxicity Measurement
• 5 The Replacement of Animal Testing: A Driving Force in Live Cell Assay Development
• 6 Regulatory Applications and Validation
• 7 Cell Signaling: At the Heart of Functional Assays for Industrial Purposes
• 8 Applications in New Drug Discovery
• 9 Impact on Health and the Environment
• 10 Outlook
• Bibliography
• Index
• End User License Agreement