Live Cell Assays: From Research to Regulatory Applications

By Christophe Furger

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.

• Language: English
• Publisher: Wiley
• Release date: Jul 14, 2016
• ISBN: 9781119330172

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Introduction

"The word cell makes us think not of a monk or a prisoner but of a bee… Who knows if the human mind, consciously borrowing the term cell from the beehive in order to designate the element of the living organism, did not also borrow, almost unconsciously, the notion of the cooperative work that produces the honeycomb?"

Georges CANGUILHEM [CAN 09]

The term live cell assay refers to all of the approaches that use the cell as an information medium for measuring purposes. This very broad definition covers a wide range of experimental contexts in which the levels of information, flow or standardization are particularly diverse and numerous. Clearly, it would be tedious to describe them all here.

For the purposes of this work, the definition will be narrowed: a live cell assay shall be defined as an approach or a technology in cell biology, which, due to its high levels of standardization, may be used by the wider scientific and industrial community for purposes of measurement and comparison. Conversely, the term application will be taken in its broader sense, covering areas as diverse as fundamental research, industrial R&D, regulatory contexts, the environment and patient diagnostics.

These applications owe their development to our capacity of manipulating the living cell and, moreover, in conserving its integrity outside of the organism. Indeed, much time was required (1910–1950) to succeed in separating (tearing would be just as appropriate) the cell from the human or animal specimen to which it belonged, and then to recognize that the isolated cell could individualize and live its own existence. From the 1960s, these developments in cell culture allowed for the emergence of the first standardized cell assays, namely, the karyotype and the Ames test.

Live cell assays are held in relatively high esteem by today’s society. We will see that this was not always the case. In so far as the models used lead to a reduction in the use of animals, cell assays are at present considered to be the more ethical choice, whether it is for research, for the discovery of new medicine or for assessing health risks.

Due to the extreme diversity of the living world, live cell assays are naturally polyvalent. They can be conceived to measure extremely specific cell activity or on the other hand, very generic activity. They can be performed on a wide array of models, from bacteria to human cells but also plants, fungi or all varieties of microorganisms. The two most common functions targeted by live cell assays are homeostasis in cases of toxicity measurements and the modulation of potential therapeutic targets in cases of pharmaceutic research. While the former represents a growing market since the introduction of international agreements such as REACH in Europe, the latter has been, for around 15 years now, a market worth several billion dollars, based in large part on the measuring of cell signaling pathways. Cyclic AMP assays remain the foremost commercial assay in terms of volume.

No reference book of which I am aware has addressed this issue in any comprehensive manner. The specialist literature remains compartmentalized, shutting itself off in various sectors such as the search for knowledge, industry, diagnostics, the environment or regulations. However, the same technologies are (or should be) at work in these different sectors. On investigation, it is clear that some of these disciplines using cell assays have not learned to communicate with each other. The objective of this work is to shed light on the contributions of these various schools of thought so as to improve the ease of exchange and, perhaps, to promote the spread of cell technology.

The development of cell assays requires knowledge of the intimate workings of the cell. Acquiring this knowledge has been particularly laborious and drawn out over several decades. A historical perspective could help to understand the importance of these works, which are seldom cited today. Indeed, historical perspective shows us the value of time. Often 20 to 30 years go by between understanding a biological mechanism and the emergence of applications that use it.

Measuring toxicity seems to have been the first application of live cell assays. In 1995, the combined contributions of molecular biology, fluorescence and genomics led to an explosion in our understanding of cellular biology, which itself led to the emergence of new generations of more varied and more informative assays in response to new requirements, particularly in the area of new drug research. These new approaches greatly benefitted from contemporary advances in miniaturization and robotization. We will see how, due to differences in schools of thought, these advances were not adopted with equal rapidity across all the sectors.

One of the major revolutions that took place in 1994 was due to the ability to substitute proteins for chemically-based fluorescent compounds. These fluorescent proteins, or GFPs, present in several species of marine animals, are coded by genes, the heterologous expression of which has been mastered. This advance unlocked vast possibilities of investigation, which naturally pushed the cell assay developers to engage without delay.

Finally, it seems appropriate to attempt this first reference book of live cell assays insofar as today we can acknowledge that the approach has acquired a certain maturity and that the applications considered are sufficiently numerous and recognized by academic, industrial and regulatory actors.

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.

Numbered-Figure

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):

– cytotoxicity measurement;

– discovery of new medicines;

– 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

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 surpass the limitations that competing tests are subject to, in terms of the predictability of effects in human beings, throughput, cost, standardization and ethical considerations, all of which may be significant for the increasingly stringent quality requirements of industrial and regulatory applications.

1.3.1. Definition and typology of cell tests

The matter of definition is fundamental. Cell biology abounds with a great many measurement methods, which have been developed in response to various issues raised over the decades. Where can a line be drawn between the cell assay and method? On what criteria should we base an assessment of the relative importance of each method? The outcome from a regulatory standpoint can be considered initially. Indeed, all tests that have followed through in implementing the organization for economic co-operation and development (OECD) guidelines or, on occasion, an ISO norm, have necessarily succeeded along the whole value chain. Nonetheless, we will see that regulatory bodies are extremely conservative and the happy few that are chosen for their list, 15 at most, are too restrictive and do not represent the diverse needs of applications.

A more reasonable criterion then is to consider the capacity of an approach to be standardized. This idea takes into account the numerous tests validated by use and not by a regulatory body. The criterion of access to high throughput will automatically permit a test to be taken into the applicative dimension and can also be retained. Several approaches that are widely practiced by the scientific community though without being standardized due to reasons of the complex process or a lack of industrial interest may also be considered as cell assays. And lastly, several approaches inspired by recently acquired developments in cell biology that are considered as fertile ground for the future of cell assays will also be brought into consideration.

The issue of cell assay typology has never truly been broached either. And this gives rise to a question: how can we rank the highly varied approaches whose only commonality is their foundation on cells in culture? The most straightforward way is to proceed according to the type of application in line with the three main areas mentioned above.

A second way to proceed is by reference to the technologies employed, which for the most part are the same in all three types of application. These technologies can be categorized into four main classes: colorimetry, fluorescence, bioluminescence and label-free methods (see Chapter 3).

A third way to address this issue is to consider the information level delivered by the approach. An assay in which the end measurement is read directly in the live cell in culture, by image analysis, for example, may be considered as more informative and pertinent than an ELISA-type test in which cells have been lysed to make the medium more homogeneous. Although found in various publications, this point of view is of debatable value since the best test is above all the one that provides the information corresponding to the question in consideration.

Finally, a last way of address the issue is to consider the status of the cell under analysis. Here we may note the following propensities, divided according to their level of complexity:

– The first consists of employing non-modified cells, or at least modified no more than least required (immortalization) for culture. In this way, cells are as close as possible to the physiological reality and may be considered to be in homeostasis. The analysis will then consist of measuring the disturbance levels of this homeostasis under the effects of a physical agent or chemical disruptor. This process finds many applications in questions of toxicity (Chapter 4) or pollution (section 9.3). In general, this approach employs colorimetric, fluorescent or bioluminescent agents, which can nonetheless disturb the signals under analysis. This problem may be avoided by means of label-free approaches that make use of a cell’s autofluorescence or of certain noninvasive electrical or optical properties (from section 3.2).

– The second consists of modifying the cell’s genome so as to transform the physical or chemical agent’s effect into a fluorescent or bioluminescent intracellular signal produced directly by the cell. Green fluorescent protein (GFP) and reporter gene strategies are typically considered to belong to this category (section 3.1).

– The third practice, and most significant in terms of activity volume, consists of verifying an independent cell function or homeostatic function, often enzymatic activity or the signaling pathway associated with a target, particularly pathological targets. Often this process requires the addition of a second genomic modification so as to create a model that independently coexpresses the luminescent signal and the target of interest. This is quite naturally put to work on the part of the pharmaceutical industry in researching new medicines. The practice will be described in Chapter 8.

All of these typologies are admissible and any preference for one or another depends only on the standpoint that actors may take within their sector of activity. The cell assays will be described here in accordance with their area of application: routine toxicity measurements (Chapter 4), regulatory toxicity measurements (end of Chapter 2, Chapter 5), researching new medicines (Chapter 8) and diagnostics (Chapter 9). The major technologies that are common to all of these various applications will be preliminarily introduced in Chapter 3.

1.3.2. The regulatory and industrial dimension

In regards to the market and access to the market, segmentation between industrial and regulatory applications becomes a necessity. Although both are engaged in live cell assays, each has developed in complete independence from the other, albeit in a parallel way. These two schools of thought were launched (or initiated) from opposite reasoning (or logic).

The main client of cell assays, the pharmaceutical industry, standardized numerous, and often highly sophisticated, cell approaches over 20 years ago in response to questions concerning the validation of therapeutic targets, the identification and validation of new compounds or toxicity measurements. More cell approaches have come to light in recent years in the area of pharmacokinetics (absorption, distribution, metabolism and excretion [ADME]), the stage preceding clinical tests. This is a significant market covering all therapeutic areas.

Regulatory authorities, on the other hand, are committed to protecting citizens from the potential dangers brought about by the industrial and agrifood industries, which every day invent and produce new substances that must be tested for their innocuousness to public health and the environment. For decades, the means employed by regulatory authorities have relied on the exclusive use of animals for their measurements. The progression of live cell assays into the market is still ongoing, and policy toward alternative approaches has remained, until recently, hesitant. Since the implementation of the 7th Amendment of the European Cosmetics Directive in Europe, banning all studies of toxicity performed on animals since 2013, the nature of the game has slowly changed. This has also been the way in the whole of the industrial sector due to the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) European Regulations. This applies to 125,000 substances, produced by the industry, whose toxicity must be tested by 2018. While the REACH regulations allow for a large part of these tests to be performed on animals, there is pressure being exerted on the official bodies that approve alternative tests to accelerate the legal availability of new approaches, in particular that of live cell assays. This pressure is even more pronounced considering that in vivo tests, aside from the ethical considerations that they raise, are adapted to neither the societal nor economic stakes in terms of both attainable throughput and cost.

It is regrettable that regulatory and industrial bodies remain so closedminded. It appears that regulatory bodies have not fully understood the advances that have been made by both the pharmaceutical industry and in academic research over these past 20 years. Or perhaps this understanding has indeed arrived, albeit very late. In any case, the lethargic pace of decision making on the part of public authorities has left legislators wishing to fulfill their obligations concerning Directives and Regulations with a relatively short list of cell assays and, more widely speaking, in vitro tests, for the most part developed in the 1970s and 1980s.

It is worth noting that the term in vitro may at times be employed to designate live cell assays. As far as regulatory organizations are concerned, the term covers all alternative methods to animal tests. In fact, most of the in vitro methods accepted as such by the official organizations are live cell assays. Some approved in vitro tests do nonetheless use extracts of human skin tissue, poultry eyes, bovine cornea or acellular biological membranes. It should also be noted that the term in silico has been accepted in reference to certain alternative methods, which, by means of software, describe the structure–function relationships (SAR) or quantitative SAR (QSAR) of compounds. These last approaches have not been validated by regulations but instead enter into certain tiered processes.

1.4. Market

Market studies constitute a burgeoning sector of activity and the global market for live cell assays is no different. Any Internet search engine will provide dozens of results on the subject at the click of the mouse.

To introduce the subject, it is worth recalling that the global market for cell assays was worth $300 million in 2002 according to a study [FRO 02] by Front Line. At that time, cell assays were considered as emergent and divided in technological terms between fluorescence (75%), bioluminescence (20%) and the remainder (nanoparticles, quantum dots, which represented <5%). Live cell tests were at the time defined as:

The tools used in analyzing the biological response of cells, specifically studies that establish the response to potential drug compounds.

It would seem that the market was directed only at the pharmaceutical industry. Their use at this time was primarily in the validation stage of targets following human genome sequencing. The massive shift toward high throughput screening had yet to take place. At that time, the factors that were associated with market growth were in connection with the discovery of new medicines, and particularly, the possibility of acquiring patents on proteins or target genes. A norm had already been established on culture in 384 well plates together with a trend toward outsourcing screening due to the cost involved and at times, due to the expertise required.

The situation is somewhat different today. A dozen studies, for the most part titled Global Cell-based Assay Market, which, according to their market evaluation in terms of volume, fall into three groups. Group 1 (low bracket) includes studies by Global Industry Analysis and Transparency Market Research, which evaluates the market at around $1.5 billion in 2017–2018. Group 2 (The Market Publisher, Companies & Markets, VisionGain, Research & Market) estimates the same market between $3.9 and 4.7 billion for 2015–2016. VisionGain expects a progression to (or: up to) $5.5 billion for 2018. The studies in group 3 (high bracket), particularly BCC Research, Markets & Markets [MAR 14], IQ4I (Intelligence Quotient for Innovation), Research & Consultancy and Market Research have announced values between $14.8 and 27 billion to be reached by 2018–2020. The significant discrepancies between these evaluations can cast doubt on their worth here, despite the studies each costing between €3,000 and 5,000. The estimate of $4.7 billion (VisionGain) is often accepted in reference articles.

According to publically released data [CLI XX], live cell assays are still an essential aspect of the pharmaceutical industry. In the sector of high throughput screening alone, live cell assays now constitute 50% of total use. This is considerable. Their versatility is appreciated. One aspect of this versatility is in allowing for an early assessment of toxicological and metabolic aspects, thereby avoiding problematic compounds before beginning highly expensive clinical trials. This is a highly desirable attribute in the eyes of industrial actors who are obsessed with knowing which candidate medicines will be defective in terms of cytotoxicity as soon as possible. The identification of such