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Animal cell cultures have been and are being used to generate valuable products based on their own genetic information or due to genes transferred into them (transgenes) using recombinant DNA technology. On the other hand, biotechnological approaches are used either to rapidly multiply animals of desired genotypes or to introduce specific alterations in their genotypes to achieve certain useful goals.
To achieve the latter more efficiently, as well as to assist in conventional breeding efforts, the entire genomes of animals are being characterised using biotechnology tools. These activities have been arbitrarily grouped under animal biotechnology since they either utilise animal cells to generate products or apply biotechnological tools to enhance the usefulness of animals to human welfare.
They are used to produce virus vaccines, as well as a variety of useful biochemical’s which are mainly high molecular weight proteins like enzymes, hormones, cellular biochemical’s like interferon’s, and immunobiological compounds including monoclonal antibodies. Animal cells are also good hosts for the expression of recombinant DNA molecules and a number of commercial products have been/are being developed.
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Initially, virus vaccines were the dominant commercial products from cell cultures, but at present monoclonal antibody production is the chief commercial activity. It is expected that recombinant proteins would become the prime product from cell cultures in the near future.
Transplantable tissues and organs are another very valuable product from ceil cultures. Artificial skins are already in use for grafting in burn and other patients, and efforts are focused on developing transplantable cartilage and other tissues.
A greater detail of cell culture products is provided under the following heads:
1. Vaccines
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2. Interferon
3. Recombinant Proteins.
Product # 1. Virus Vaccines:
A vaccine is a preparation containing a pathogen (disease producing organism) either in attenuated or inactivated state. This preparation is introduced into an individual to induce adequate antibody production against the pathogen in question so that the individual becomes protected against infection, at a later date, by that pathogen. The introduction of a vaccine in an individual is called vaccination or immunization as it leads to the development of immunity in the vaccinated individuals to the concerned pathogen.
The immunity is induced by the antigens of pathogen origin and present in the vaccine. Any molecule that induces production of antibodies specific to itself when introduced in the body of an animal is called antigen. Usually the antigenic function is confined to a rather small portion of the antigen molecules; such a region is known as antigenic determinant or epitope.
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A B-lymphocyte cell becomes predetermined, during differentiation, to produce antibodies of a single specificity. Ordinarily in an animal, the frequency of B-lymphocytes capable of producing antibodies of a given specificity, i.e., against a single epitope, is very low. (However, the B-lymphocyte population of an animal produces antibodies against an amazingly large number of epitopes or antigenic determinants).
When an antigen enters into the body of an animal, its antigenic determinant ultimately binds to the specific receptors present on the surface of those B-lymphocytes which produce antibodies specific to this antigen.
This binding stimulates a rapid proliferation of these B-lymphocytes so that the proportion of such cells increases drastically; this is called clonal selection (proposed by N. Jerne). As a consequence, the concentration in the blood serum of antibodies (antibody titre) specific to the concerned antigen also increases dramatically. This phenomenon is the basis of immunisation.
When an individual is vaccinated, the antigens of pathogen origin stimulate antibody production against themselves. This increase in antibody production takes some time, but since the vaccine does not have virulent pathogens there is no danger of disease development.
(If the pathogen present in the vaccine were virulent/alive, vaccination would produce disease before the antibody production could be sufficiently increased). When live virulent form of the same pathogen later enters into the system of an immunised animal, the high level of antibodies specific to the pathogen (present in the animal as a result of immunisation) inactivate the pathogen, and thereby protect the animal against the disease.
The various vaccines can be grouped into two categories:
i. Vaccines containing killed or inactivated pathogenes, i.e., most bacteria vaccines and some virus vaccines'(e.g., influenza virus inactivated by formalin, rabies virus inactivated by phenol or Propiolactone), and
ii. Those containing live but attenuated pathogens, e.g., most virus vaccines.
Attenuation means a drastic reduction in the virulence of a pathogen; this is achieved as follows:
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a. Several consecutive passages through an animal which is not the usual host of the pathogen, e.g., small pox virus in calf.
b. Several passages through cultured cells of the host, e.g., rabies virus in human diploid cell culture, or of a different species, e.g., rabies virus, yellow fever virus in chick embryo cell culture.
c. Selection of less virulent strains of pathogens, e.g., a mutant strain of polio virus.
d. Treatment of the pathogen with some chemicals, e.g., B.C.G. (Bacillus of Calmette Guerien) vaccine produced by culturing the bacteria on a medium containing bile.
e. Culturing pathogens under unfavorable conditions like high temperature, e.g., anthrax vaccine obtained by cultivation of the bacterium (Bacillus anthracis) at 40°-50°C.
In general, inactivation of viruses is always coupled with attenuation to minimise the accidental presence in vaccines of active virulent particles which could cause disease in the vaccinated individuals.
The different vaccines differ in their composition, efficacy and the duration of effective protection to the vaccinated individuals. Vaccines are one of the earliest examples of biotechnological intervention in human and animal health care.
Vaccines offer the cheapest and most effective protection against diseases, and for some diseases, e.g., hepatitis B, AIDS etc., they are the only means of protection. The effectiveness of vaccines may be highlighted by the success of WHO (World Health Organisation) sponsored mass vaccination against small pox in completely wiping out this once ravaging disease from the face of earth.
The procedure of virus vaccine production using cell cultures is essentially and in simple terms as follows. The cells to be used as host are inoculated in culture vessels and, after suitable growth has occurred; the cultures are infected with the virus concerned and incubated.
After an appropriate period of virus multiplication and release, the culture medium containing virus particles is collected and suitably processed. In case of killed virus vaccines, the vaccine is usually subjected to a concentration step after virus inactivation. Suitable stabilizers are added to prevent loss of potency and the vaccines are generally stored at low temperature till used. A rigorous quality control is maintained throughout the entire operation.
A number of vaccines are produced in cultured animal cells; some of these are being produced in India but the procedure has several inherent problems.
These are:
(i) Rigorous quality control is essential particularly to exclude the possibility of presence of infectious agents in the materials and cells used. In addition,
(ii) the vaccine may contain a rare virulent and active virus particle which may cause disease in the vaccinated individuals.
Therefore, alternative approaches of vaccine production and delivery have been/are being developed, viz.:
(i) Production of antigenic proteins or only their antigenic determinants in GEMS (genetically engineered microbes).
(ii) Use of DNA as vaccines
(iii) Production of antigenic proteins in vegetables/fruits to minimise storage costs and problems.
Product # 2. Interferon’s:
Interferon’s are proteins produced by a cell infected by a virus, and provide protection to other healthy cells from viruses. Interferon was discovered in 1957 when Issacs and Lindenmann observed that virus- free fluid obtained from cultured cells infected with virus protected other cells from virus infection. They called the substance present in these fluids, which interfered with virus infection, interferon.
There are three major types of interferons:
(i) Interferon-α (INF-a; produced by leucocytes or white blood cells).
(ii) Interferon-β (INF-p; produced by fibroblasts).
(iii) interferon-y (INF-y; produced by stimulated T- lymphocyte cells, hence also called immune interferon).
The mechanism of protection by interferons appears to be as follows. When interferon reacts with the interferon receptors of a cell, the cell enters in a state called interferon-induced antiviral state. In this state, a rapid degradation of mRNA occurs if the cell is infected by any virus.
Interferon induces in cells the production of 2, 5-adenosine polymerase. When such a cell is infected by a virus, the 2, 5-A polymerase is activated to produce 2, 5 adenosine (2, 5-A), which in turn activates pre-existing but inactive molecules of ribonuclease-L. Activated ribonuclease-L degrades all mRNA (of host as well as virus origin) present in the cell bringing the protein synthesis to a halt in such cells.
This interferes with multiplication of the virus so that virus infection is either stopped or sufficiently slowed down to allow the production of adequate antibodies against the invading virus. The protection due to interferons is nonspecific in that interferon induced by any one virus will provide protection against all viruses.
It is possible that interferons modify ribosomes so that they no longer translate viral mRNAs, although they are fully capable of translating mRNAs of the host origin. Interferons enhance the cytotoxic activity of natural killer cells (NK cells), which are a type of lymphocytes identifiable as large granular lymphocytes (LGL). NK cells are cytotoxic to some types of tumour cells. Interferons are known to inhibit growth of some types of tumours; most of these tumors are also responsive to chemotherapy.
Interferons, therefore, have been employed in treatment of the responsive tumors despite their prohibitive cost (initially, $50 million/ g of commercial product). Interferons are produced from human leucocytes isolated from donor blood and cultured in vitro, and from mouse fibroblast cultures. The production scheme, in simple terms, is as follows. Large scale cell cultures are infected with Sendai virus, and incubated for 24 h after which the supernatant (clear fluid) is collected, centrifuged, and used for interferon isolation.
The amount of interferon recovered is relatively small (1 g interferon of low purity from leucocytes separated from blood of about 90,000 donors), and the normal leucocytes are difficult to culture preventing scaling up from relatively small inocula. These contributed to the enormous price of the product.
In view of the value of and demand for interferons, intensive efforts were made to produce it in genetically engineered organisms, e.g., E. coli, yeast mammalian cell cultures and even in plants These efforts have drastically reduced the cost (to less than 10% of the initial price) and improved the purity (by several orders of magnitude) of the product.
Product # 3. Recombinant Proteins:
Proteins produced by genes transferred into selected host cells by genetic engineering are called recombinant proteins since they are based on recombinant DNA technology. Recombinant proteins form an important component of biopharmaceuticals, i.e., biotechnology products having pharmaceutical applications.
A large number (nearly 2 dozen) of recombinant proteins are being produced in mammalian cell cultures some of which, viz., human growth hormone (HGH), tissue plasminogen activator (tPA), erythropoietin and blood clotting factor VIII, are already in therapeutic use. The host cells used for large scale production of the various recombinant proteins are: Chinese hamster ovary line (CHO), baby hamster kidney line BHK, mouse mammary tumour line C127, mouse myeloma cell lines and mammalian cell lines.