APPLICATIONS OF ANIMAL CELL CULTURE
The animal cell cultures are used for a diverse range of research and development. These areas are:
a) production of antiviral vaccines, which requires the standardization of cell lines for the multiplication and assay of viruses.
b) Cancer research, which requires the study of uncontrolled cell division in cultures.
c) Cell fusion techniques.
d) Genetic manipulation, which is easy to carry out in cells or organ cultures.
e) Production of monoclonal antibodies requires cell lines in culture.
f) Production of pharmaceutical drugs using cell lines.
g) Chromosome analysis of cells derived from womb.
h) Study of the effects of toxins and pollutants using cell lines.
i) Use of artificial skin.
j) Study the function of the nerve cells.
Somatic Cell Fusion
One of the applications of animal cell culture is the production of hybrid cells by the fusion of different cell types. These hybrid cells are used for a the following purposes:
(i) study of the control of gene expression and differentiation,
(ii) study of the problem of ‘ malignancy’,
(iii) viral application,
(iv) gene mapping,
(v) production of hybridomas for antibody production.
In 1960s, in France for the first time, the hybrid cells were successfully produced from mixed cultures of two different cell lines of mouse. Cells growing in culture are induced by some of the viruses such as ‘Sendai virus’ to fuse and form hybrids. This virus induces two different cells first to form heterokaryons. During mitosis, chromosomes of heterokaryon move towards the two poles and later on fuse to form hybrids. It is important to remove the surface carbohydrates to bring about cell fusion. Some other chemicals like polyethylene glycol also induce somatic cell fusion.
Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated.
Fig showing the production of t-PA
Tissue Plasminogen activator (t-PA)
was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.
Blood Factor VIII
Haemophilia A is a blood disorder which is a sex-linked genetic disease in humans. The patients suffering from Haemophilia A lack factor VIII, which plays an important role in the clotting of blood. This factor VIII is secreted by a gene present on X-chromosome but this gene undergoes mutations in people suffering from Haemophilia. Current therapy for this disease is the transfusion of blood factor VIII into patients. Using rDNA technology, Factor VIII has been produced from mammalian cell culture e.g. Hamster kidney cell.
The EPO is a glycoprotein consisting of 165 amino acids and is formed in the foetal liver and kidneys of the adults. It causes proliferation and differentiation of progenitor cells into the erythrocytes (erythroblasts) in the bone marrow. Erythropoietin is hormone-like in nature and is released by the kidney under hypoxic or anoxic conditions caused by anaemia.
Amgen Inc. holds US patent for preparation of, eErythropoietin, by recombinant method using Chinese Hamster Ovary cell lines. Erythropoietin (EPO) is a hormone-like substance released by the kidney under hypoxic or anoxic conditions caused by anaemia. r-HUEPO- recombinant human erythro- protein has been effectively used to treat anemia associated with AIDS, renal failure etc.
The production of Monoclonal Antibodies using hybridoma technology
Antibodies are proteins synthesized in blood against antigens and are collected from the blood serum. The antibodies, which are heterogenous and non specific in action are called polyclonal antibodies. If a specific lymphocyte, after isolation and culture in vitro becomes capable of producing a single type of antibody bearing specificity against specific antigen, it is known as monoclonal antibody. The monoclonal antibodies are used in the diagnosis of diseases because of the presence of desired immunity. However, these antibody secreting cells cannot be maintained in culture. It was observed that the myeloma cells (bone marrow tumour cells due to cancer) grow indefinitely and also produce immunoglobulins which are infact monoclonal antibodies.
In 1974, George Kohler and Milstein isolated clones of cells from the fusion of two parental cell lines – lymphocytes from spleen of mice immunized with red blood cells from sheep and myeloma cells. These cells were maintained in vitro and produced antibodies. The hybrid cells maintained the character of lymphocytes to secrete the antibodies, and of myeloma cells to multiply in culture. These hybrid cell lines are called “Hybridoma” and are capable of producing unlimited supply of antibodies. Hybridoma are obtained by using an antibody producing lymphocytes cell and a single myeloma cell. Monoclonal antibodies bind very specifically to an epitope (specific domains) on an antigen and by using them it is possible to detect the presence of specific antigens.
The Monoclonal antibodies are used for the treatment of patients with malignant leukaemia cells, B cell lymphomas and allograft rejection after transplantation. CD3 is an antigen present on the surface of mature T- cells lymphocytes. If T- cell population is depleted or controlled, the transplanted organ will not be rejected. An antibody that acts against CD3 surface antigen of T-cells is called OKT3 i.e. anti-CD3 Moab. OKT3 is a monoclonal antibody which has been licensed for clinical use for the treatment of acute renal allograft rejection. OKT3 removes antigen bearing cells from circulation thereby helps in accepting the graft.
Fig showing the steps involved in the production of monoclonal antibodies
When Monoclonal antibodies are used as enzymes using the technique of enzyme engineering, then they are called abzymes.
Using animal cell cultures, it is also possible to produce Polyclonal Antibodies. Polyclonal antisera are derived from many cells therefore contains heterogeneous antibodies that are specific for several epitopes or an antigen.
SCALE-UP OF ANIMAL CELL CULTURE
Modifying a laboratory procedure, so that it can be used on an industrial scale is called scaling up. Laboratory procedures are normally scaled up via intermediate models of increasing size. The larger the plant, the greater the running costs, as skilled people are required to monitor and maintain the machinery.The first pre-requisite for any large scale cell culture system and its scaling up is the establishment of a cell bank. Master cell banks (MCB) are first established and they are used to develop Master Working Cell Banks (MWCB). The MWCB should be sufficient to feed the production system at a particular scale for the predicted life of the product. The cell stability is an important criteria so MWCB needs to be repeatedly subcultured and each generation should be checked for changes. A close attention should be paid to the volume of cultured cells as the volume should be large enough to produce a product in amounts which is economically viable. The volume is maintained by a) increasing the culture volume, (b) by increasing the concentration of cells in a reactor by continuous perfusion of fresh medium, so that the cells keep on increasing in number without the dilution of the medium.
A fully automated bioreactor maintains the physicochemical and biological factors to optimum level and maintains the cells in suspension medium. The most suitable bioreactor used is a compact-loop bioreactor consisting of marine impellers. The animal cells unlike bacterial cells, grow very slowly. The main carbon and energy sources are glucose and glutamine. Lactate and ammonia are their metabolic products that affect growth and productivity of cells. So, the on-line monitoring of glucose, glutamate, and ammonia is carried out by on line flow injection analysis (FIA) using gas chromatography (GC), high performance liquid chromatography (HPLC) etc.
In batch cultures, mainly Roller Bottles with Micro Carrier Beads (for adherent cells) and spinner flasks (for suspension cultures) are used in Scale-up of animal cell culture process.
The Roller bottles provide total curved surface area of the micro carrier beads for growth. The continuous rotation of the bottles in the CO2 incubators helps to provide medium to the entire cell monolayer in culture.The roller bottles are well attached inside a specialized CO2 incubators. The attachments rotate the bottles along the long axis which helps to expose the entire cell monolayer to the medium during the one full rotation. This system has the advantage over the static monolayer culture: (a) it provides increase in the surface area, (b) provides constant gentle agitation of the medium, (c) provides increased ratio of surface area of medium to its volume, which allows gas exchange at an increased rate through the thin film of the medium over the cells. Typically, a surface area of 750-1500 cm2 with 200-500 ml medium will yield 1-2x108cells.
Diagram showing the Roller bottle cell culture
Micro Carrier Beads
Micro carrier beads are small spherical particles with diameter 90-300 micrometers, made up of dextran or glass. Micro Carrier beads, increase the number of adherent cells per flask. These dextran or glass-based beads come in a range of densities and sizes. The cells grow at a very high density which rapidly exhausts the medium and therefore the medium has to be replaced for the optimum cell growth. At the recommended concentration when the microcarriers are suspended they provide 0.24 m2 area for every 100 ml of culture flask.
The spinner flask, was originally developed to provide the gentle stirring of microcarriers but are now used for scaling up the production of suspension cells. The flat surface glass flask is fitted with a Teflon paddle that continuously turns and agitates the medium. This stirring of the medium improves gas exchange in the cells in culture. The spinner flask used at commercial scale consists of one or more side arms for taking out samples and decantation as well.