Photo: GSK Marburg

Beneficial Effects of Animal Cell Technology

(last update: 1998)

About this Publication

Scientific research is not only dedicated to increase the wealth of human knowledge, but is committed to apply that knowledge for the benefit of nature in general and the human race in particular. The rapid increase in medical know-how during the last fifty years has saved millions of lives that otherwise would have been lost. Indeed, many future generations will exist which consequently would not have done so due to the associated extinction of the invidividual genetic information.

These rapid improvements in health care have been strongly influenced by discoveries from modern methods of biotechnology. Animal cell technolgy plays a substantial role in the field of biotechnology and may be defined as ‘the use of animal cells propagated in-vitro (that is, outside the animal usually in so called bioreactors) for the manufacture of bioproducts and as vehicles in the discovery and/or the testing of medicines’. This technology is now widespread in modern pharmaceutical research.

The Animal Cell Technology Industrial Platform (ACTIP) is an association of European companies actively involved in the use of animal cell technology. This publication has been compiled by scientists representing major European industries.

Presently, research in animal cell technology is taking place in or is being supported by industry to a larger extent than in the past. Discoveries leading to further progress, therefore, come from both industry as well as academic organisations. The purpose of this publication is to express ACTIP’s viewpoints regarding the contribution of animal cell technology to society by giving an overview of the past, present and future of animal cell technology, its potential and its limitations. Data is presented here covering the entire span of industrial applications, continuing developments and research topics.

Based on our knowledge and experience, we are convinced that animal cell technology as a part of the modern ‘biotechnologies’, is and will remain a meaningful tool in the development and production of new vaccines as well as therapeutic and diagnostic products. It can contribute to improved quality of life of both animals and humans and should result in a reduction in the number of animals used when developing health products.

Contents

1. Introduction

1.1 The History of Animal Cell Technology

1.2 New Technologies Boost Applicability

1.3 Why Animal Cells and not Other Cells?

2. Applications of Animal Cell Technology

2.1 Clinical Applications

2.2 Preservation of the Environment: Ecology in Production

2.3 Reduced Use of Animals

2.4 Applications in Research

3. Future Developments in Animal Cell Technology

3.1. Basis for Medical Treatments

3.2 Vaccines

3.3 In-Vitro Models for Toxicology

4. The Future: Input from Industry and Society

Glossary of Terms

 

1. Introduction

1.1 The History of Animal Cell Technology

For thousands of years, mankind has benefited from biological processes in daily life: the baking of bread, the brewing of beer and the fermentation of milk or cabbage to preserve foods are only a few examples. At the beginning of this century, the word biotechnology was coined in the milk industry. Since then it has come to mean the practical application of biological processes for technical production. Animal cell technology, sometimes referred to as cytotechnology, is a part of biotechnology. The propagation of animal cells in-vitro (outside the animal) allows the large-scale production of a range of bioproducts. Animal cell technology can be defined as:

“that part of biotechnology which uses animal cells, propagated in-vitro, for the manufacture of bioproducts and as tools in the discovery and testing of medicines”

Animal cell technology decreases considerably the total number of animals used for preventive, therapeutic or diagnostic purposes and provides a large potential to develop useful products.

1.1.1 Beginning of Animal Cell Technology: Vaccines

In 1797 the British physician Edward Jenner observed that milkmaids did not develop smallpox because they had been frequently exposed to cow pox. He then laid the foundation for the science of immunology in the Western world by injecting a child with the fluid from the nodules caused by the cow pox virus to protect the child against smallpox. In the decades following, based on the scientific work of the French chemist Louis Pasteur, it became apparent that exposure to a small amount of a modified or attenuated infectious agent created immunity for some diseases. As of 1883, the first children were being vaccinated against rabies and a new industry, the vaccine industry, was born.

In order to expand the range of protective vaccines, a vigorous search for the agents responsible for various infectious diseases and for methods to attenuate their virulence was initiated. Viruses and bacteria were often found to be the culprits. This work was carried out by scientists in academic institutions as well as by scientists in the emerging vaccine industry, which sought to

isolate the starting material for a vaccine, e.g. the responsible bacteria and viruses. In the early days the vaccine industry used animals such as artificially infected rabbits for rabies vaccine or cows for vaccines against smallpox as a source of some of the required viruses for viral vaccines. They also used bacteria or toxins as the basis for bacterial vaccines. Between 1920 and 1950, virus and bacterial vaccines against diseases such as typhoid, diptheria, tuberculosis, tetanus, cholera, pertussis, influenza and yellow fever were developed and introduced.

Culturing cells to produce viruses
In the early 1950s techniques became available to culture animal cells for the production of viruses. This involves the large-scale cultivation of animal cells in a specialized vessel containing a specific nutrient medium. The end result will be that viruses are produced in the cells. This breakthrough was the real start of animal cell technology or cytotechnology.

Animal cell technology-based virus vaccines may contain live but harmless variants of the infectious agent, called attenuated viruses, or they may contain killed viruses. These early applications of animal cell technology have saved millions of human and animal lives over the past 30 years. Between 1950 and 1985, cytotechnology and progress in other techniques led to the introduction of human vaccines against such diseases as polio, measles, mumps, rubella, hepatitis B and varicella zoster. Numerous veterinary vaccines were also developed.

From primary cells to continuous cell lines
In the early days of animal cell technology, primary cell cultures were used. To produce a vaccine against the polio virus, for example, cells were taken from a monkey kidney, kept in culture for some days and infected with the virus in order to propagate more virus. To increase production, the number of animals was simply increased. Since animal cell technology developed rapidly as the preferred method to produce viral vaccines, developments took place to increase the efficiency of the process and to decrease the use of animals needed for a cell culture. A real breakthrough was the development of continuous cell lines; cells are taken from an animal and propagated in-vitro under specific conditions to greatly expand the number of cells available for virus production. Occasionally some cell clones develop which can grow indefinitely. These cells are cultured to establish ‘cell banks’ consisting of many aliquots of the same cell type. The need to return to animals to replenish cells is thereby eliminated. Cell banking and other developments have saved the lives of countless numbers of animals whose organs and lives

might otherwise have been necessary for the production of the required viruses. Even more important was the effect on quality. Because of the use of highly characterised master cell banks, which are rigorously tested for safety, greater consistency in originating cells is achieved. The variability seen between animals was eliminated and the potential for accidentally introducing infectious agents from animals was greatly reduced.

1.1.2 Technology Suitable for the Production of Natural Compounds

Limited use for protein production
Using similar cell culture techniques it became possible to produce other natural compounds, predominantly proteins. A prerequisite was the ability to obtain homogeneous populations of cells with the capability to synthesise the desired protein. In these systems, however, the production rate was usually low and the costs and effort often exceeded the benefit. This considerably limited the usefulness of animal cell technology for production purposes, other than for the production of vaccines or a very limited number of proteins such as interferons and urokinase. With the advancement of science, however, this situation changed in the 1970s.

1.2 New Technologies Boost Applicability

1.2.1 Hybridoma Technique

Specific antibodies in quality and quantity
A major turning point expanding the industrial applicability of animal cell technology occurred with two scientific revolutions in the early 1970s. One was the establishment of the hybridoma technique. This describes a method for fusing or ‘hybridising’ cells which have the ability to produce specific antibodies with other types of cells that allow the hybrid cell or ‘hybridoma’ to be cultivated indefinitely. With this technique it became possible to produce specific antibodies with consistent quality in very large quantities previously only dreamed of.

1.2.2 Recombinant-DNA Technology

Protein production in sufficient quantities
The other accelerator and facilitator in animal cell technology was the technique which we today call ‘genetic engineering’ or ‘recombinant-DNA technology’. This technology allows the genetic information coding for proteins of interest to be inserted into cells which lend themselves to propagation on an industrial scale. The technology enables the efficient production of many proteins which were previously unavailable or not available in sufficient quantities.

Genetic engineering also makes it possible to produce proteins which have been modified or tailored to increase their utility. Because of gene transfer techniques, it is also possible to choose for production purposes cell lines which are well characterised, have a history of safe and efficient use and are suited for industrial processing.

1.2.3 Products Based on New Technologies

Production of new substances
The hybridoma technique and recombinant-DNA technology boosted the use of animal cell technology for the production of vaccines and particularly its use for the production of natural therapeutic or diagnostic compounds. Today the list of products resulting from these methods is extensive (see Table ). It comprises drugs for the treatment of cardiovascular diseases (tissue plasminogen activator: tPA), cystic fibrosis (DNases), anemia (erythropoietin: EPO), hemophilia (coagulation factors VIII and IX), cancer and viral infections (interferons) and dwarfism (human growth hormone: hGH). The list also contains many monoclonal antibodies used as diagnostics and many more are in late phase clinical trials as a therapeutic agent. Increasingly, vaccines are also produced using genetically modified animal cells. These examples demonstrate the tremendous impact of animal cell technology on the improvement of animal and human health.

1.3 Why Animal Cells and not Other Cells?

When one has chosen a particular cell line as starting material for the application of recombinant-DNA technology (e.g. a cell line >from a hamster ovary or a cell line from a monkey kidney), this cell line is called the host system. The host systems we are talking about in this brochure come from mammals or insects, hence animal cells. For some applications systems might be based on other hosts such as bacteria, yeasts or filamentous fungi.

1.3.1 Host Systems Compared

Many host systems available
For the expression of some products host organisms other than animal cells have been evaluated: bacteria (Escherichia coli, Bacillus subtilis), yeast (Saccharomyces cerevisiae, similar to baker’s yeast), streptomycetes and filamentous fungi.

Product type determines choice
All these systems have their advantages and disadvantages. Whilst micro-organisms are easier to cultivate than animal cells, the latter have some useful characteristics. In higher organisms many modifications of a synthesised protein occur between the translation of the DNA-code into a protein and its secretion into the cell’s environment. These changes, called post-translational modifications, are often necessary for the biological activity of the product. Recombinant animal cells are able to modify and secrete proteins in the same way as it happens in the original sources. In microbial systems, in contrast, these modifications are seldom performed completely or correctly

Microbial systems: small, simple proteins
Microbial systems tend to be used for relative small, simple proteins without extensive posttranslational modifications, e.g. insulin. Animal cell systems are mainly used for large, complex proteins that require posttranslational modifications for their bioactivity, such as tissue-plasmiogen activator (tPA) or erythropoietin (EPO).

Animal cell systems: large, complex proteins
In some cases the production of a substance is possible in either an animal cell system or a microbial system. For example, human growth hormone and interferon-alpha, which do not carry any posttranslational modification, can therefore be produced in either animal cells or micro-organisms. The manufacturer’s considerations such as production costs or ease of purification will determine the choice of systems. A summary of advantages and disadvantages of the various host organisms is shown in Table 2.

Table 2.

General characteristics of various expression systems for the production of recombinant proteins.

Yeast and filamentout fungi Bacilli E. coli Animal Cells
Authenticity of secondary product structure (folding and S-S bridge formation) varying varying varying yes
Removal of N-terminal methionine yes varying varying yes
Secretion varying varying rarely yes
Protein modification
e.g. glycosylation
varying, but different from mammalian cells no no yes
Fermentation time days hours hours week(s)

 

 

1.3.2 The Potential of Animal Cell Cultures

Nowadays, animal cell technology has advanced to the stage where it is used in a variety of manufacturing processes. Although the technology will be further improved, there are already several products derived from this technology on the market. In fact, products derived from animal cell cultures already generate more than half of the revenues from the sale of modern biotechnology products. More importantly, many of the products of animal cell technology were previously not available in sufficient quantities for therapy or could only be produced at prohibitive costs or with extreme difficulty. In the next sections we will review the benefits of products obtained from animal cell technology.

2. Applications of Animal Cell Culture

2.1 Clinical Applications

2.1.1 Safety for Patients

One of the most convincing advances achieved by animal cell culture is increased safety of products.

In the past accidents resulting from unwanted allergic or pathogenic substances found in animal-derived substances have occurred. SV40 contaminations of polio vaccines, influenza vaccines contaminated with allergenic hen’s egg proteins, growth hormone preparations from human pituitaries causing Creutzfeldt-Jakob disease and blood products infected with the Hepatitis virus (HBV) or Human Immunodeficiency Virus (HIV) are some of the better known examples.

Safer products

Although it is impossible to give a 100% guarantee that accidents will not occur in the future, our worldwide experience with this technology has taught us that these can be minimized by better defining contaminating agents and by improving production processes. The use of animal cell technology has been a major step forward towards safer products. Employing animal cell technology the risk of contamination with pathogenic agents is reduced because well-characterised cell lines are used. Below we will give some examples of how safety issues have been addressed.

Safer vaccines

Vaccines were originally produced by propagating the agents in animals. The dangers inherent to this process are obvious; other pathogens from the donor organism may be transmitted by this procedure. Today, viruses can be propagated in well-characterised cell lines. This as well as extensive purification steps greatly reduce the risk of contaminating pathogens.

Only virus-specific proteins

An even more sophisticated technology uses genetic engineering to produce defined viral protein sequences in well-characterised cell cultures. These protein sequences are then used to immunize humans against the disease in question. This technology eliminates the need to handle whole, infectious viruses.

Non-infected products

Examples can also be found in the area of therapeutic products. Some therapeutics (for example, factor VIII and IX, which are used in the treatment of hemophilia as discussed below) have been obtained until til now by extraction from blood of voluntary donors. Because of this, there has been the potential for contamination with viruses causing human diseases such as hepatitis B to be transferred to patients receiving treatment with these products. The transfer of HIV, causing AIDS, through infected blood products is another example. The use of genetically modified animal cells has allowed the production of much safer and non- infected products such as the coagulation factors VIII and IX, which were formerly exclusively extracted from blood sources. Thus, the risk of many infections has been greatly reduced.

2.1.2 Quantity and Quality of Available Therapeutics

Quantities sufficient for treatment

Many therapeutics would never have been available for treatment of patients because they could only be found in or extracted >from natural sources in minimal quantities. Now, however, if one knows the gene sequence coding for a protein, cells can be supplied with the exclusive genetic information to produce the desired therapeutic molecule. The cells are cultured to provide large quantities of the product of interest. This development is a major thrust in the animal cell technology industry. For instance, there is continuous development of new cytokines or cytokine-antagonists, products not available from human sources. Below we look at some examples that are already on the market or under development.

Sufficient and safe human growth hormone

Human growth hormone, which is used for the treatment of dwarfism, was extracted from the pituitary glands of deceased humans before it could be obtained from genetically modified animal cells. To produce the amount of growth hormone necessary to treat one patient, tens to hundreds of human pituitaries had to be processed. The quantity produced was only sufficient to treat some, but not all, patients. In addition, this production method involved a risk of transferring an infectious agent causing Creutzfeldt-Jakob disease. The current production method using genetically modified animal cells enables the treatment to be offered to all patients. In addition, it is a much safer product. A recombinant human growth hormone produced in a genetically engineered bacterium is also available.

Enabling treatment of anemia resulting from kidney disease

Another example is erythropoietin. Formerly, 2,500 litres of urine from patients with aplastic anemia had to be processed to obtain minute quantities of erythropoietin for laboratory analyses. It was impossible to establish a production process before the advent of recombinant-DNA technology in animal cell cultures. Now, with the aid of genetically modified cells, it is possible to produce sufficient erythropoietin to treat many thousands of patients with anemia resulting from kidney disease.

Blood clot dissolving agent available

Another product that could not be previously made in sufficient quantities to treat patients is tissue-plasminogen activator (tPA), a product used for the conditions of pulmonary embolism and myocardial infarction. Only the biotechnological production of recombinant tPA in animal cell cultures yields sufficient quantities of tPA for clinical treatment of patients. This recombinant tPA is made from the same human gene as the natural tPA produced in our body. So far more than 500,000 patients worldwide have been treated with recombinant tPA. In different multicenter clinical trials it has been clearly demonstrated that the clinical impact of this type of thrombolytic therapy is significant. The benefits for the patients are higher survival rates and higher reperfusion. There are less side effects compared to other thrombolytic agents or invasive methods. At present the clinical treatment of lung embolisms and deep vein occlusions (thromboses) using tPA are under investigation.

Alternative source for blood coagulation factors

Many patients with hemophilia are treated with Factor VIII or Factor IX. Traditionally, these factors are produced from plasma. This source, however, is in short supply as a consequence of the increased testing and quality demands on donor’s blood in light of the potential danger from AIDS, hepatitis and unknown viruses. The production of factors VIII and IX using animal cell technology will ensure sufficient virus-free supplies.

In addition, some of the patients treated with conventional Factor VIII or Factor IX products have developed inhibitors against these drugs. In principle another coagulation factor, Factor VII could be used for treatment of these patients. Unfortunately, Factor VII is available only in minute quantities in body fluids. It could therefore not be isolated from blood samples in quantities sufficient for treatment of all hemophiliacs with inhibitors. The only way to make Factor VII available for therapeutic application is via modern biotechnology: synthesis on the basis of recombinant-DNA technology in a mammalian cell line. Factor VII produced in animal cells is expected to reach the market soon.

2.1.3 Human-like Products

Prevention of immune reactions

Non-human proteins of animal origin that are used for therapeutic purposes can be recognised by the human immune system as ‘foreign proteins’. This may induce immune reactions. After the first application, every further use may increasingly cause severe immune reactions.There are many historical examples to illustrate this drawback of non-human products. These include: streptokinase, a bacterial protein used to treat myocardial infarction; antibodies from animals e.g. against tetanus toxin; and insulin extracted from pigs, found to be antigenic in some diabetic patients.

Genetic engineering helps to overcome the problem of immune reactions. Either by expressing the authentic human genes in animal cells or by replacing parts of animal genes by human gene sequences (e.g ‘humanising’ of mouse antibody molecules), human-like or human-identical molecules can be obtained from non-human sources. Using this technique, the occurrence of allergic reactions can be reduced and repeated or continuous use of the same drug becomes feasible. Below we look at some examples illustrating this important development.

Human-identical lymphokines

Lymphokines, including the interferons, are produced using animal cell technology and genetic engineering.

Interferons are effectively used in the treatment of cancer and virus diseases.

In nature, they are only found in very low amounts in human tissue. In the body their normal task is related to regulation of cell metabolism, proliferation and differentiation. For human therapy, they are used mainly for the treatment of certain types of cancer.

Lymphokines exhibit species specificity. In humans, only the human lymphokines are active. Thus, the use of animal organs for the production of lymphokines is not possible because the non-human lymphokines would be inactive. It was also impossible to try to isolate lymphokines from human tissue for therapeutic purposes – too much human tissue would be needed for processing to help even one patient!

Various solutions have been found to circumvent this problem. For example, some interferons are made by culturing cells which naturally make interferons. It is also possible to introduce human genes coding for lymphokines into well-characterised animal cell lines, which may then produce lymphokines in large quantities. Some interferons are also made successfully in bacteria. Because the genetic code is identical to a human lymphokine, the lymphokines produced closely resemble their naturally occurring human counterparts.

2.1.4 New Products

New products

With animal cell technology it is possible to develop new products. Some of these products are already on the market; others are still under development. Below two groups of such new compounds, monoclonal antibodies and fusion proteins, are presented.

Monoclonal antibodies

With cell culture consistent quality and quantity

Monoclonal antibodies are antibodies with a single specificity. They can be produced by fusing a specific antibody-producing cell, which has a limited lifespan, with a cell type that does not produce antibodies but has infinite cell division capability. The result of such a fusion is a cell line with the potential for continuous propagation and continuous production of pure antibodies in large quantities.

Monoclonal antibodies can be produced in-vivo as mouse ascites tumours or in-vitro by culturing the fused cells. The in-vivo method can result in the sacrifice of large numbers of animals and presents the risk of contaminating the product with infectious agents from the mice. These disadvantages are overcome by producing monoclonal antibodies in cell culture.

Numerous diagnostic applications

Because of their specificity, monoclonal antibodies are well suited as diagnostic agents for laboratory tests. In fact, diagnostic products based on monoclonal antibodies were the first products of the ‘new biotechnologies’ to enter the market. At present, hundreds of such products are available. Their application ranges from detecting pregnancy to blood group typing prior to blood transfusion as well as detection of hormone deficiencies.

Making diseased tissues visible

An elegant approach to directly identify abnormal tissues in patients is the use of monoclonal antibodies that are coupled to an image-giving molecule. Because of the specificity of the antibody in this complex, it can bind specifically to, for example, receptors on tumour cells or on certain cells in a thrombus (blood clot). Since an image-giving molecule, for example a radioactive isotope, is also present in this complex, their location in the body can be visualised with the appropriate equipment. Next to diagnostics of cancer or thrombosis, this technique allows in-vivo diagnostics of many cell types or tissues involved in various diseases.

Applications where specificity is needed

The applications of monoclonal antibodies as diagnostic agents are not limited to the field of health care. They can also be used in agriculture and used as a tool in quality control. They can even be used to simplify and improve specificity of purification steps in a variety of industrial processes. They can, for instance, extract specifically the desired product from a ‘broth’ containing a range of proteins.

Monoclonal antibodies as therapeutics

Today, monoclonal antibodies are already widely acknowledged as diagnostics and production tools. Their therapeutic application is just starting and many applications are being evaluated in the clinic. Developments with respect to therapeutic monoclonal antibodies have already progressed far in the treatment of cancer, AIDS and other diseases. Some products are already on the market, e.g. monoclonal antibodies for the treatment of transplant rejection and for the diagnosis of some cancers.

Magic bullets: concentration of ‘killing compounds’ where needed

Another example of the therapeutic use of monoclonal antibodies produced with animal cell cultures is the ‘magic bullet’ concept, which uses a radioisotope, a toxin or a protoxin-activating enzyme coupled to a specific antibody. The specific antibody recognises certain antigens on the surface of e.g. a tumour cell. Because of the specificity of the antibody, the complex binds only to the surface of the tumour cells. The radioisotope or toxin present in the complex is thereby specifically concentrated around the tumour cells. In the case of a protoxin-activating enzyme coupled to a monoclonal antibody, the patient is treated first with the enzyme-complex and then with the protoxin. The protoxin is activated by the enzyme, releasing a toxin. This toxin is located on the tumour cells because the enzyme is coupled to the specific antibody which binds to and concentrates on the surface of the tumour cells. This latter approach has the advantage that one can delay the administration of the protoxin until the antibody- enzyme conjugate that has not bound has been cleared from the body. This way, the toxin will not exert its effect in other parts of the body and the local effect of the toxin will be enhanced.

Fusion proteins

Fusion proteins as a new product concept

Another example where animal cell technology has led to a novel product concept is the production of fusion molecules. Here, the properties of different molecules are combined first at the genetic level to obtain improved final products. One example is the fusion of a gene coding for an immune response enhancing human cytokine to one of the genes coding for a component of a monoclonal antibody directed against a cancer cell specific antigen. After expression of the complete modified monoclonal antibody (i.e. the antibody fused to the cytokine) in animal cells, it can be purified and injected into cancer patients to augment the body’s own immune response specifically against the tumor cells, because the cytokine in the fusion protein is concentrated at the site of the tumor. The validity of this concept has still to be proven. However, this type of approach is likely to be used in similar treatments of other diseases.

2.2 Preservation of the Environment: Ecology in Production

Harmless, natural materials

The use of animal cell technology has little impact on the environment. The materials used in the manufacturing process are generally harmless natural materials. For example, the major components of the nutrient media used to cultivate animal cells are salt, sugar, vitamins and amino acids. These components do not present any harm to the environment.

Small scale

For most applications the dosage required for patient treatment is small. As a consequence, on a yearly basis relatively small amounts are needed for diagnosis or treatment of patients. Therefore, most of the production processes using animal cells take place on a relatively small scale compared with many conventional pharmaceuticals. Because of the small scale, small amounts of process water and energy are consumed.

Purification advantages

Also the chemicals used for purification are usually not harmful, because this would destroy the product to be purified.

Purifying a compound from a relatively pure and well defined source such as a cell culture is much simpler than the purification >from a rather undefined source such as, for example, animal tissue.

Record of safety

Finally, a word on safety. The vaccine industry pioneered high standards of safety and learned to produce under safe conditions for its workers and the environment. This experience laid the foundation for current work with animal cells. Such cells, including recombinant cells, have been safely used in academic and industrial laboratories for many years. On production scale our experience is increasing. All this work is subject to strict regulation by government authorities to ensure safety for man and the environment.

2.3 Reduced Use of Animals

2.3.1 Antibody Production

Before animal cell technology offered alternatives (see 2.1.4), antibodies were produced in large amounts by injecting animals with the antigen, the substance which will elicit the immune response. After the animal showed the immune reaction, the antibody was purified from its blood. Besides the ethical barrier against this process, this production method holds several disadvantages. First of all, the batch-wise production of antibodies in animals is subject to the naturally occurring variability inherent to the use of animals. A second disadvantage is that the use of live animals always carries the risk of potential contamination with unknown pathogens. The third disadvantage is the limited possibility to increase the production scale. When only milligram or gram quantities of the product are required, the use of live animals might be justified. However, it would require enormous numbers of animals to satisfy the need for several kilograms of a therapeutic product.

Production of monoclonal antibodies in bioreactors

Animal cell technology for the production of monoclonal antibodies offers a solution. A few mice or other antibody-producing animals have to be initially immunised to provide the antibody-producing B-cells. These cells are harvested from the animal and subsequently combined or fused with cells which have continuing division capability. The result is hybridoma cells which are immortal and produce the monoclonal antibody. They can be grown in-vitro in a bioreactor any time the antibody is needed. Thus, for subsequent project development and production, there is no further need for animals – the animals can be saved and replaced by bioreactors.

2.3.2 Drug Development

Cell cultures for early drug screening

Experimental animals are used in the search for new, safe and efficient drugs. Potential drug candidates are presently screened in animal models. Drugs earmarked for further development are tested again for possible toxic effects in animals before they are tested in humans. The number of animals needed in early drug screening can now be drastically reduced using animal cell culture models. Cell models help the scientists to understand function and mechanism of the diseases under study. Interaction of therapeutics with their specific targets can thus be studied before their application. For complete characterisation and safety testing of compounds identified in animal cell culture screening systems, experiments with living animals will, however, still be necessary. This is needed because complex processes such as the pharmacokinetics and pharmacodynamics of potential drug candidates or the systemic toxicological effects need to be studied in whole organisms. Only these exhibit the large variety of interactions and reactions which reflect the situation in the human body as closely as possible.

New techniques are being developed continuously to improve the properties of animal cell cultures as early drug screening models. In section 2.4. representative examples of these screening models are given.

2.4 Applications in Research

The previous chapters and sections mainly described the use of animal cell cultures for production purposes. In this section we focus on its use in research. Many exciting avenues of study have been opened by the technique of genetically modifying animal cells intended for culture. Unfortunately, the examples described below require some basic knowledge of biology to fully understand the potential of these techniques. This section, therefore, is mainly intended for the reader with some knowledge of the field.

2.4.1 Structure of Therapeutics and Other Proteins

Sufficient quantities for structure studies

In order to understand the mechanism underlying the action of a drug on the molecular level, one has to understand a drug’s structure. With knowledge of a molecule’s structure, it may be possible to establish its mode of action, its interaction, and other effects.

Proteins and peptides are usually rather large and complex molecules. These proteins and peptides play a major role in the regulation of cell development, cell-to-cell-communication, signal transduction mechanisms within tissues, immune responses and many other basics of our biology and existence. So, how can important structural information about these large and complex molecules be obtained? Their three dimensional structure can be elucidated using physical methods such as crystallography and X-ray structure analysis. To do so, the substance to be studied has to be crystallised and, for that purpose, high concentrations of the substance are needed. Obtaining sufficient quantities of the protein or peptide to be crystallised was in the past a severe problem. Today, however, in many cases the necessary amounts can be obtained if the molecules can be produced in cell culture.

The molecular structures of erythropoietin, tissue plasminogen activator (tPA) and the protease from HIV have been solved by crystallography. Cell cultures enabled the scientists to obtain sufficient material for the crystallisation. For example, the structure of human growth hormone has been studied by many groups to determine the mechanism for receptor-ligand binding. The blood coagulation factor VIII is also under structural scrutiny, mainly to study the structure-function relationship.

2.4.2 Screening Models for New Compounds

Receptors do the screening

Many diseases are due to deficiencies of hormones or other metabolic regulators. Thus, for therapeutic purposes, such hormones or other regulators need to be available. The action of hormones and regulators involves receptors at a cell surface to which the hormone or regulator is bound. If the structure and genes of a particular receptor are known, it is possible, with genetic modification, to manipulate specific cells in such a way that they will express such a receptor on their surface or in their cytoplasm. These modified cells can then be grown in culture and used in the search for drugs with receptor-binding properties. Only those drug candidates selected by this screening procedure have to be developed in further tests on authentic tissues.

More reliable results

These types of screening systems are, for instance, used in the search for new anxiolytics, which are drugs to reduce anxiety in depressed people and for the treatment of insomnia.

In such studies neurotransmitter receptors can be expressed on the surface of recombinant-DNA modified animal cells and the binding and action of new drug candidates can be studied. Models are usually more reliable than experiments with whole animals because the receptor-carrying cells can be grown with a high consistency and the response will not be influenced by other reactions. Animals sometimes respond with individual variances, which hinders the interpretation of the results. Thus, because of improved clarity and higher reliability of the cell culture model, fewer experiments are needed to produce significant results.

Human receptors for species-specific results

Original human cells or cell lines can also be used in the search for promising new compounds. This offers the advantage that the receptor is of the human type and not of another species. If a new compound binds to a human receptor, there is a high probability that the compound will exert effects in humans. This way, species-specificity problems which are often encountered with the animal screening systems can be prevented. However, quite often specific receptors are expressed at a very low level or are found in such an environment that the results obtained are difficult to interpret. More studies are therefore needed to develop these models further.

Similar screening models can be developed by introducing human receptors in non-human animal cells. It is possible to actually build human receptors into cell membranes of cultured

animal cells providing a ‘chimeric’ model. Thereby, the real effect of a drug on human receptors can be studied in-vitro. This is a very important development, since in-vivo models are always based on the use of a non-human species. The results obtained with in-vivo animal models may not be the results one hopes to eventually see in human tissue. To circumvent the use of irrelevant non-human models and the need for original human tissue, the development of chimeric models is very important because the interaction between a human receptor and a potential drug candidate can be portrayed.

2.4.3 Complex Models for Functional Screening and Drug Metabolism Studies

Co-expression for functional information

Extremely exciting is the potential to genetically modify cultured cells by introducing more than only the receptor to be studied. This is done by so-called co-expression of a receptor-linked reporter system. In contrast to the systems described above, not only ligand binding can be studied, but also the functional effect of a modulated receptor on gene regulation. With this method even more relevant results can be obtained. If a certain receptor is coupled to the appropriate transduction mechanism, only those drugs which induce the entire signal cascade will elicit increased expression of the reporter gene, leading to an easily detectable indicator signal. An example of this approach is the simultaneous expression of a steroid-hormone activated receptor together with the hormone-responsive DNA region coupled to a reporter gene. Drugs proving their efficacy in such cell culture models are used for further development and studies. Despite all initial technical intricacies, this model is easy to use in the search for agonists or antagonists for a specific hormone, since the activity of the reporter gene is modulated directly.

Drug metabolism studied in-vitro

Another example for the successful use of animal cell culture using genetically modified cells is the area of in-vitro pharmacology. Genetically modified animal cells expressing an enzyme catalysing only a single step in the metabolism of a drug molecule can be used as a tool for pharmacokinetic studies. The highly specific products will not degrade in these recombinant cells, which were selected for very low endogenous metabolic activity when not stimulated.

3. Future Developments in Animal Cell Technology

3.1 Basis for Medical Treatments

The previous sections dealt primarily with the use of animal cell technology for production or research purposes. It is expected that animal cell technology will continue to contribute to the availability of new vaccines, diagnostic and therapeutic products and research tools. We will not review in detail future developments in these fields, because many opportunities have previously been mentioned.

Cell culture basis for medical treatment

Animal cell technology is also the basis for some exciting new developments in the medical field. When looking at the future of animal cell technology, we therefore first look at these prospects, most notably somatic gene therapy or viral vector therapy and the use of cell cultures for artificial organs. We conclude this section with a review of developments in the fields of vaccines and in-vitro toxicity testing.

3.1.1 Viral Vector Therapy

Viruses bring genes into the cell

Animal cell cultures are the basis for numerous clinical trials for the treatment of diseases involving either genetic defects or cancer. These trials deal with gene therapy of somatic cells or viral vector therapy. This involves the production, using animal cell culture, of genetically modified viruses which contain the gene for a missing function or for additional genetic information. These viruses do not replicate in the patients but transfer the ‘healthy’ gene code to somatic cells as the central part of therapy.

As of March 1994, approximately 60 protocols for clinical phase I trials using these methods had been approved worldwide. More than 200 patients were involved in these studies. These trials addressed such diseases as cystic fibrosis, Duchenne’s muscular dystrophy and forms of cancer, including melanoma.

Although the procedures for gene therapy are still in their infancy, promising results have been obtained. It is expected that the impact of gene therapy on many so far untreatable diseases will be large.

3.1.2 Artificial Organs

Cells grow to form skin

Another exciting development involving cell culture technology concerns the cultivation of organs or tissue in-vitro.Today it is already possible to grow skin in culture and to use it as replacement of a patient’s skin. This method has already been successfully applied in severely burned patients. The technique first involves taking a patient’s own cells. These are brought into culture and grown on a tissue-compatible support. While growing, they reassemble into a skin-like tissue. This tissue is then used to replace the patient’s burned skin. The recovery achieved with artificial ‘own tissue’ is much more satisfactory than the one resulting from the use of skin from donors or artificial skin replacements. Fewer immune reactions are seen because the tissue is highly compatible with the skin of the recipient.

3.1.3 Somatic Cell Therapy

Implanting cells to restore function

It is not only possible to culture in-vitro complete organs such as skin but various human tissues having a specific function might be grown as well. For instance, it has been considered of developing alternative treatments of diabetes by implanting islet cell tissue, first grown in culture, into the pancreas of a patient. Another application under consideration is the implantation of kidney cells for patients with kidney insufficiencies or the growing of brain cells for patients with Parkinson’s disease. Finally, in several hospitals in Europe and the USA, phase I trials have been started involving injection of a patient with transfected cells. These transfected cells secrete anti-tumour substances or factors stimulating a patient’s immune response (such as TNF or interferon). These applications, which still require much further development before being realised, are called somatic cell therapy.

3.1.4 Anti-Cancer Cells

Patient receives own, activated cells

The future might well see applications of cell therapy in medicine. It has already been possible to extract macrophages and other cells from a patient’s immune system.These extracted cells were primed in-vitro to specifically attack tumour cells. Afterwards, these activated immune cells were reintroduced into the patient’s circulation. They were then able to recognise and attack exclusively cancer cells, including the ones metastasizing and circulating in the blood stream. Thus, these activated immune cells were directing the breakdown of the tumour cells.

3.2 Vaccines

As indicated in the introductory chapter, most viral vaccines find their origin in animal cell technology. They are either produced conventionally by propagating vaccine strains of the different viruses in animal cell cultures or by recombinant-DNA technology.

Empty virus particles for vaccines

A new development in the vaccine area is the production of intact empty virus particles. Such empty virus particles consist of all structural viral proteins which elicit the immune response but they lack viral nucleic acids. They, therefore, lack any infectivity. In addition, vaccines made with such empty particles will have a superior immunogenicity in comparison with vaccines consisting of a single viral protein, because of the perfect presentation of the viral epitopes and their long serum half-life. For example, such empty virus particles could be produced with genetically modified recombinant baculoviruses produced in cultured insect cells because the genes for several epitopes may be expressed simultaneously by baculoviruses.

Deleting genes for safe vaccines

Another line of research in the virus vaccine area involves deletion of a gene or part of a gene which codes for the pathogenic properties of a virus. Such targeted mutations give rise to attenuated virus strains which are much safer than existing attenuated virus strains obtained by conventional attenuation methods. These latter strains have mutated only in a few base pairs in the genetic code. Therefore, they always present the risk that they might revert into virulent wild strains. The deleted-gene approach is now being pursued, for example, in the search for vaccines against AIDS.

One vaccine prevents many diseases

Another line of research in the vaccine area is the use of live vectors, such as genetically modified vaccinia viruses cultured in animal cells. These vaccinia viruses might be constructed in such a way that they express on their surface the antigenic determinants of several different pathogens. Immunisation with a vaccine containing such a single, multi-purpose vaccinia virus might then give protection against various infectious agents whose antigenic determinants have been inserted into the vaccinia virus. This opens exciting prospects for a single-shot, multi-purpose vaccination. A similar approach can be envisaged using other vectors such as e.g. Lactobacteria. This experimental approach is being followed for vaccines against rabies, hepatitis B and malaria.

Antibody functions as antigen

As a last example of new developments in the area of vaccines produced with cultured animal cells, the use of anti-idiotypic antibodies as antigens should be mentioned. This approach starts with making an antibody against an antigen; then a second antibody against the first antibody is made, hence the name anti-idiotypic antibody. The second antibody, because of its ‘fit’ to the first antibody, mimics the original antigen.Thus, the anti-idiotypic antibody functions as an antigen and can be used instead of the original antigen to provoke a similar, specific immune response. The anti-idiotypic antibodies are non-infectious. They are proteins, not carrying any infectious RNA or DNA and, therefore, much safer to use. This approach is being pursued for a vaccine against hepatitis B.

As in all other cases, the optimal strategy for the design of a new vaccine must be individually considered for each pathogen or group of pathogens. Thus, the future might bring sub-unit vaccines, empty-particle vaccines, multi-purpose vaccines and anti- idiotypic vaccines. This demonstrates how powerful the new techniques are, bringing an extremely wide range of opportunities to the field of animal cell technology.

3.3 In-Vitro Models for Toxicology

Reliable, safe and reproducible tests

In earlier sections it was mentioned how cell cultures are used as screening models in the search for new drugs and other products. This is not only done to obtain superior, consistent results or to speed up the drug selection process. A worthwhile goal is reduction of animals needed for drug screening.

When drugs or other products coming into human contact are being developed, their potential toxicity needs to be established. In fact, many toxicity tests are required by the authorities before a product can be marketed. Most of these toxicity tests involve the use of animals. At present, a massive effort is underway to develop reliable, safe and reproducible in-vitro toxicity tests based on animal cell culture technology rather than using whole animals. However, for the foreseeable future, animal testing will probably not be completely eliminated, since complex systemic toxicology studies can only give meaningful results if the response in a whole animal is obtained. The cell culture models can, however, further help to reduce the number of animals used.

The Future: Input from Industry and Society

In animal cell technology developments have gone beyond the goal of obtaining new products for research, diagnostics, therapeutics and vaccines. New applications, such as gene and cell therapy, are evolving. Industry has provided and still provides tools to realise new applications. It is up to society, however, to discuss the value and benefits of these applications, the ethical implications and to establish rules to prevent misuse of drugs or technologies. We in industry are willing to provide society with the information needed and we welcome discussion of these issues. As industry we consider it important to:

  1. work with national and international regulatory agencies toensure that products are safe, efficacious and are not misused;
  2. build up efficient control measures;
  3. speak openly about our products and applications;
  4. inform society in an understandable way about current developments and existing products.
Glossary of Terms

Animal cell technology: that part of biotechnology which uses animal cells, propagated in-vitro, for the manufacture of bioproducts and as tools in the discovery and testing of medicines.

Antigen: any substance which is capable of inducing a specific immune response and of reacting with the products of that response (e.g. an antibody or specifically sensitised T-cells).

Antigenic determinant: the structural component of an antigen molecule that is responsible for specific interaction with antibody molecules elicited by the same or a related antigen.

Ascites: the accumulation of serous fluid in the abdominal cavity of a mammal.

Attenuation: decreasing the virulence of a pathogenic organism.

Bioreactor: a closed vessel for the cultivation of cells under controlled conditions.

Cell bank: a uniform pool of cells, distributed into vials and preserved, typically by freezing in liquid nitrogen. A vial is thawed to start each new production cycle.

Cell line: cells which result from the subcultivations of primary cell cultures.

Continuous cell line: a cell line which has acquired the characteristic of being ‘immortal’, i.e. capable of growing indefinitely in culture.

Epitope: an antigenic determinant of known structure.

Expression: the formation of proteins and peptides in a cell or on a cell surface by translation of the genetic code.

Gene: the biological unit of heredity, containing information for structure and function. A gene is composed of a specific sequence of nucleic acids.

Genetic engineering: a term which comprises methods for (1) the isolation and recombination of genetic material (including recombination of DNA from different species), (2) the insertion of recombinant DNA into an organism, either the original one or one in which this DNA does not naturally occur (host cell) and (3) the replication and expression of this DNA in its new environment.

Gene therapy: modification of genetic information of somatic cells of the patient with the purpose to replace or supplement defective or missing parts of cellular DNA.

Host system: a cell type which is used as the carrier for DNA from another source.

Humanising: adaptation of non-human genes to the human genetic code, to enable animal cells or bacteria to express human-like molecules.

Hybridoma: a cell line resulting from the fusion of an antibody secreting blood cell (lymphocyte) and an immortal myeloma cell line. Produces a ‘monoclonal’ antibody.

Immunoglobulin: a protein of animal origin endowed with known antibody activity, synthesised by lymphocytes and plasma cells. They function as specific antibodies and are responsible for the humoral (blood-related) aspects of immunity.

Immunology: that branch of medical science concerned with the response of the organism to antigenic challenge, the recognition of self from non-self, and all the biological, serological and physical-chemical aspects of immune phenomena.

Lymhokines: a ‘family’ of proteins produced by lymphocytes (blood cells) which have a wide range of regulatory functions within the body.

Magic bullet: the concept describing the linking of a specific antibody (often a monoclonal antibody) to a toxin or drug with the purpose of bringing the toxin or drug specifically to the target cell or tissue.

Medium: fluid composed of all nutrients necessary for cell cultivation.

Nutrients: substances necessary for growth, normal functioning and maintaining life. Most often amino acids, carbohydrates, minerals, fatty acids and vitamins.

Peptide: a class of compounds of low molecular weight which consist of two or more amino acids.

Pharmacodynamics: the biochemical and physiological effects of drugs and the mechanisms of their actions.

Pharmacokinetics: the reaction of the body on a drug over a period of time, including processes of absorption, distribution, localisation in tissues, biotransformation and excretion.

Posttranslational modification: changes to a protein occurring after the genetic code has been ‘translated’ into an amino acid sequence (protein). Examples are the folding of a protein, the secondary deletion of amino acids, the formation of structural bridges or the addition of small molecules (sugar, phosphates etc) to a protein.

Primary cell culture: a culture of cells, tissues or organs taken directly from organisms and before the first subculture.

Protein: large complex organic compounds built of many amino acids. They are the principle constituents of cells.

Receptor: a specific molecule on the surface or within the cytoplasm of a cell that specifically recognises and binds other molecules, e.g. antigens, hormones or neurotransmitter molecules. The specific binding elicits a specific response (reaction).

Recombinant-DNA technology: see genetic engineering.

Somatic cell therapy: the administration of cells grown or treated in culture to a patient to cure or alleviate the symptoms of a disease.

Systemic toxicity: damaging effects to the body as a whole.

Transgenic organism: an organism living with an altered or additional gene as a result of genetic engineering.

Transfection: the process of transferring or introducing a gene or nucleic acid to a recipient cell.

 

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