ANIMAL CELL CULTURE

INTRODUCTION

An important aspect of any biotechnological processes is the culture of animal cells in artificial media. These animal cells in culture are used in recombinant DNA technology, genetic manipulations and in a variety of industrial processes. Now-a -days it has become possible to use the cell and tissue culture in the areas of research which have a potential for economic value and commercialization. The animal cell cultures are being extensively used in production of vaccines, monoclonal antibodies, pharmaceutical drugs, cancer research, genetic manipulations etc.Animal cells e.g. egg cells are used for multiplication of superior livestock using a variety of techniques like cloning of superior embryonic cells, transformation of cultured cells leading to the production of transgenic animals. The animal cells are also usedin vitrofertilization and transfer of embryos to surrogate mothers. Hence the establishment and maintenance of a proper animal culture is the first step towards using them as tools for biotechnology.

History :

1878: Claude Bernard proposed that physiological systems of an organism can be maintained in a living system after the death of an organism.1885: Roux maintained embryonic chick cells in a saline culture.1897: Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma.1903: Jolly observed cell division of salamander leucocytes in vitro.1907: Harrison cultivated frog nerve cells in a lymph clot held by the 'hanging drop' method and observed the growth of nerve fibers in vitro for several weeks. He was considered by some as the father of cell culture.1910: Burrows succeeded in long term cultivation of chicken embryo cell in plasma clots. He made detailed observation of mitosis.1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth.1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods.1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells.1923: Carrel and Baker developed 'Carrel' or T-flask as the first specifically designed cell culture vessel. They employed microscopic evaluation of cells in culture.1927: Carrel and Rivera produced the first viral vaccine - Vaccinia.1933: Gey developed the roller tube technique.1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture.1948: Earle isolated mouse L fibroblasts which formed clones from single cells. Fischer developed a chemically defined medium, CMRL 1066.1949: Enders reported that polio virus could be grown on human embryonic cells in culture.1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells. Dulbecco developed plaque assay for animal viruses using confluent monolayers of cultured cells.1954: Abercrombie observed contact inhibition: motility of diploid cells in monolayer culture ceases when contact is made with adjacent cells.1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium.1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed that they have a finite lifespan in culture.1964: Littlefield introduced the HAT medium for cell selection.1965: Ham introduced the first serum-free medium which was able to support the growth of some cells.1965: Harris and Watkins were able to fuse human and mouse cells by the use of a virus.1975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody.1978: Sato established the basis for the development of serum-free media from cocktails of hormones and growth factors.1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent.1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use.1986: Lymphoblastoid IFN licensed.1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available.1989: Recombinant erythropoietin in trial.1990: Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2).

Animal cell culture media :Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions. Glucose is added as energy source-its concentration varying depending on the requirement. Phenol Red is added as a pH indicator of the medium.There are two types of media used for culture of animal cells and tissues- the natural media and the synthesized media.Natural Media - The natural media are the natural sources of nutrient sufficient for growth and proliferation of animal cells and tissues. The Natural Media used to promote cell growth fall in three categories.Coagulant, such as plasma clots. It is now commercially available in the form of liquid plasma kept in silicon ampoules or lyophilized plasma. Plasma can also be prepared in the laboratory taking out blood from male fowl and adding heparin to prevent blood coagulation.Biological fluids such as serum. Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum is obtained from human adult blood, placental, cord blood, horse blood, calf blood. The other forms of biological fluids used are coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes etc.Tissue extracts for example Embryo extracts-Extracts from tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow etc are also used for culture of animal cells.Synthetic media - Synthetic media are prepared artificially by adding several organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors etc. Different types of synthetic media can be prepared for a variety of cells and tissues to be cultured. Synthetic media are of two types- Serum containing media (media containing serum) and serum- free media (media without serum). Examples of some media are: minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12 etc.Advantages of serum in culture medium are:i) serum binds and neutralizes toxins,(ii) serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins,(iii) it contains the protease inhibitors,(iv) it increases the buffering capacity,(v) it provides trace elements.Disadvantages of serum in culture medium are:(i) it is not chemically defined and therefore its composition varies a lot,(ii) it is sometimes source of contamination by viruses, mycoplasma, prions etc,(iii) it increases the difficulties and cost of down stream processing,(iv) it is the most expensive component of the culture medium. pH- Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth. The optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular enzymes and binding of hormones and growth factors to cell surface receptors in the cell cultures. The regulation of pH is done using a variety of buffering systems. Most media use a bicarbonate-CO2 system as its major component.Osmolality- A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in the growth media determine the osmolality of the medium. All commercial media are formulated in such a way that their final osmolality is around 300 mOsm.

Primary cell culture, Subculture, Monolayer and Suspension Culture :The maintenance of growth of cells dissociated from the parental tissue (such as kidney, liver) using the mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers is called Primary Cell Culture.The primary cell culture could be of two types depending upon the kind of cells in culture.

a) Anchorage Dependent /Adherent cells- Cells shown to require attachment for growth are set to be Anchorage Dependent cells. The Adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. They grow adhering to the cell culture.

b) Suspension Culture/Anchorage Independent cells- Cells which do not require attachment for growth or do not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes.Secondary cell culturesWhen a primary culture is sub-cultured, it becomes known as secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel.Subculturing- Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the growth media, washing the plate, disassociating the adhered cells, usually enzymatically. Such cultures may be called secondary cultures.

Development of cell lines:A Cell Line or Cell Strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines are categorized into two types:a)Finite cell Lines- The cell lines which have a limited life span and go through a limited number of cell generations (usually 20-80 population doublings) are known as Finite cell lines. These cell lines exhibit the property of contact inhibition, density limitation and anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours.b)Continuous Cell Lines- Cell lines transformed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines. The cell lines show the property of ploidy (aneupliody or heteroploidy), absence of contact inhibition and anchorage dependence. They grow in monolayer or suspension form. The growth rate is rapid and doubling time is 12-24 hours.c)Monolayer cultures- When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures.d)Suspension cultures- Majority of continuous cell lines grow as monolayers. Some of the cells which are non-adhesive e.g. cells of leukemia or certain cells which can be mechanically kept in suspension, can be propagated in suspension. There are certain advantages in propagation of cells by suspension culture method.These advantages are:(a) The process of propagation is much faster.,(b) The frequent replacement of the medium is not required.,(c) Suspension cultures have a short lag period,(d) treatment with trypsin is not required,(e) a homogenous suspension of cells is obtained,(f) the maintenance of suspension cultures is easy and bulk production of the cells is easily achieved.,(g) scale-up is also very convenient.The cell lines are known by:a) A code e.g. NHB for Normal Human Brain.b) A cell line number- This is applicable when several cell lines are derived from the same cell culture source e.g. NHB1, NHB2.c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two doublings.

Growth KineticsThe standard format of a culture cycle beginning with a lag phase, proceeding through the logarithmic phase to a stationary phase and finally to the decline and death of cells, is welldocumented. Although cell growth usually implies increase in cell numbers, increase in cell mass can occur without any replication. The difference in mean cell mass between cellpopulations is considerable, as would be expected, but so is the variation within the same population.Growth (increase in cell numbers or mass) can be defined in the following terms.a. Specific growth rate, jjl (i.e. the rate of growth per unit amount (weight/ numbers) of biomass): \l = (1/x) (dx/dtjh-1 3Where dx is the increase in cell mass, dt is the time interval, and x is the cell mass. If the growth rate is constant (e.g. during logarithmic growth), then In x = In xq -t- |xt 4 where x0 is the biomass at time t0.b. Doubling time, td (i.e. the time for a population to double in number/mass):In 2 0.693 td - Vc.Degree of multiplication, n or number of doublings (i.e. the number of times the inoculum has replicated): n = 3.32 log(x/Xo) 6

Types of culture methods :Organ culture and Histotypic culturesThe cell-cell interaction leads to a multistep events in in vivo situations. For example, hormone stimulation of fibroblasts is responsible for the release of surfactant by the lung alveolar cells. Androgen binding to stomal cells stimulates the prostrate epithelium. In other words, hormones, nutritional factors and xenobiotics exert stimulating effects on the cells to function in a coordinated manner. Xenobiotics broadly refers to the unnatural, foreign, and synthetic chemicals such as pesticides, herbicides, refrigents, solvents and other organic compounds. It is impossible to study these cellular interactions that occur in the in vivo system with isolated cells or cells in culture. This has lead to the attempts to develop organ and histotypic culture with the aim of creating in vitro models comparable to the in vivo system. The three types of such cultures are:a) Organ culture- In this type of culture, the whole organs or small fragments of the organs with their special and intrinsic properties intact are used in culture.b) Histotypic culture- The cell lines grown in three dimensional matrix to high density represent histotypic cultures.c) Organotypic cultures- A component of an organ is created by using cells from different lineages in proper ratio and spatial relationship under laboratory conditions.Organ cultureIn the organ culture, the cells are integrated as a single unit which helps to retain the cell to cell interactions found in the native tissues or organs. Due to the preservation of structural integrity of the original tissue, the associated cells continue to exchange signals through cell adhesion or communications. Due to the lack of a vascular system in the organ culture, the nutrient supply and gas exchange of the cells become limited. In order to overcome this problem, the organ cultures are placed at the interface between the liquid and gaseous phases. Sometimes, the cells are exposed to high O2 concentration which may also lead to oxygen induced toxicity. Due to the inadequate supply of the nutrients and oxygen, some degree of necrosis at the central part of the organ may occur. In general, the organ cultures donot grow except some amount of proliferation that may occur on the outer cell layers.Techniques and Procedure for organ cultureIn order to optimize the nutrient and gas exchanges, the tissues are kept at gas limited interface using the support material which ranges from semisolid gel of agar, clotted plasma, micropore filter, lens paper, or strips of Perspex or plexiglass. The organ cultures can also be grown on top of a stainless steel grid. Another popular choice for growing organ cultures is the filter-well inserts. Filter-well inserts with different materials like ceramic, collagen, nitrocellulose are now commercially available. Filter well inserts have been successfully used to develop functionally integrated thyroid epithelium, stratified epidermis, intestinal epithelium, and renal epithelium.The procedure for organ cultures has the following steps:a) The organ tissue is collected after the dissection.b) The size of the tissue is reduced to less than 1mm in thickness.

c) The tissue is placed on a gas medium interface support.d) Incubation in a CO2 incubator.e) M199 or CMRL 1066 medium is used and changed frequently.f) The techniques of histology, autoradiography, and immunochemistry are used to study the organ cultures.The advantages of organ cultureThe organ cultures can be used to study the behavior of an integrated tissue in the laboratory. It provides an opportunity to understand the biochemical and molecular functions of an organ/tissue.Limitations of organ cultureIt is a difficult and expensive technique. The variations are high with low reproducibility. For each experiment, a new or fresh organ is needed as organ cultures are not propagated.Histotypic culturesUsing histotypic culture, it is possible to use dispersed monolayers to regenerate tissue like structures. It the growth and propagation of cell lines in three-dimensional matrix to high cell density that contributes to this. The techniques used in histotypic cultures are:a) Gel and sponge technique- In this method, the gel (collagen) or sponges (gelatin) are used which provides the matrix for the morphogenesis and cell growth. The cells penetrate these gels and sponges while growing.b) Hollow fibers technique- In this method, hollow fibers are used which helps in more efficient nutrient and gas exchange. In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed to be used for histotypic type of cultures. The cells get attached to capillary fibers and increase in cell density to form tissue like structures.c) Spheroids The re-association of dissociated cultured cells leads to the formation of cluster of cells called spheroids. It is similar to the reassembling of embryonic cells into specialized structures. The principle followed in spheroid cultures is that the cells in heterotypic or homotypic aggregates have the ability to sort themselves out and form groups which form tissue like architecture. However, there is a limitation of diffusion of nutrients and gases in these cultures.d) Multicellular tumour spheroids- These are used as an in vitro proliferating models for studies on tumour cells. The multicellular tumour spheroids have a three dimensional structure which helps in performing experimental studies related to drug therapy, penetration of drugs besides using them for studying regulation of cell proliferation, immune response, cell death, and invasion and gene therapy. A size bigger than 500 mm leads to the development of necrosis at the centre of the MCTS. The monolayer of cells or aggregated tumour is treated with trypsin to obtain a single cell suspension. The cell suspension is inoculated into the medium in magnetic stirrer flasks or roller tubes. After 3-5 days, aggregates of cells representing spheroids are formed. Spheroid growth is quantified by measuring their diameters regularly. The spheroids are used for many purposes. They are used as models for a vascular tumour growth. They are used to study gene expression in a three-dimensional configuration of cells. They are also used to study the effect of cytotoxic drugs, antibodies, radionucleotides, and the spread of certain diseases like rheumatoid arthritis.Organotypic culturesThese cultures are used to develop certain tissues or tissue models for example skin equivalents have been created by culturing dermis, epidermis and intervening layer of collagen simultaneously. Similarly models have been developed for prostrate, breast etc. Organotypic culture involves the combination of cells in a specific ratio to create a component of an organ.Stem Cell Culture:Stem cells are the raw material from which all of the bodys mature, differentiated cells are made. Stem cells give rise to brain cells, nerve cells, heart cells, pancreatic cells, etc. They have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses.They can replicate themselves over and over for a very long time.Understanding how stem cells develop into healthy and diseased cells will assist the search for cures.There are two types of Stem CellsEmbryonic (also called pluripotent) stem cellsEmbryonic stem cellsare capable of developing into all the cell types of the body. There are two Sources of Embryonic Stem Cells-1) Excess fertilized eggs from IVF (in-vitro fertilization) clinics can be used as a source of embryonic stem cells. Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment. These surplus embryos can be used to produce stem cells. Regenerative medical research is finding modern methodology to develop these cells into new, healthy tissue to treat severe and often fatal illnesses.2) Therapeutic Cloning (Somatic Cell Nuclear Transfer)In the Somatic Cell Nuclear Transfer, the nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example). No sperm is involved in this process, and the embryo are not created to be implanted in a womans uterus. The resulting stem cells can be induced to develop into specialized cells that are useful to treat dangerous illnesses.

Adult stem cellsAdult stem cellsare less versatile and more difficult to identify, isolate, and purify.The stem cells are extracted from a 5-7 days old blastocyst. Stem cells can divide in culture to form more of their own kind,thereby creating a stem cell line. Later these are induced to generate healthy tissue needed by patients.The Importance of Stem Cell TherapyStem cells allow us to study how organisms grow and develop over time. Stem cells can replace diseased or damaged cells that cannot heal or renew themselves. To develop and research and find new drugs and medicines, stem cells can be used to test these chemical and drugs. Stem cells can helps us to understand what is called the genetic machinery. All ready tremendous efforts are going on to treat diseases like Parkinsons Disease, Leukemia (Bone Marrow Transplants) diabetes, multiple sclerosis, etc using stem cell therapy. Another area of importance is to regenerate tissues to be used as skin grafts to treat patients suffering severe burns.TISSUE ENGINEERING

Tissue engineering refers to the application of the principles of engineering to cell culture for the construction of functional anatomical units- tissues/organs. The aim of tissue engineering is nothing but to supply the various body parts for the repair or replacement of damaged tissues or organs. It is now possible to grow skin cells, blood cells cardiac cells etc. by using the ability of stem cells to proliferate and differentiate.During the last decade, the tissue culture work in animals demonstrated that virtually any human tissue or organ can be grown in culture. This became possible only after it became known that the ability of cultured cells to undergo differentiation can be restored. Skin was the first organ to be cultured in artificial media and could be successfully used for transplantation following serious skin burns. For past few years some of the biotech companies like ATS (Advanced Tissue Science, USA), Biosurface Technology (BTI, Cambridge) and Organogenesis, are developing artificial skins to the stage of clinical trials.In the field of tissue replacement, focus of attention is the Artificial cartilage. As it is not vascularized, it is not rejected due to immunogenic response. This will have lots of implications in the treatment of sport related injuries and diseases like arthritis.Design and engineering of tissuesThe design and tissue engineering should essentially cause minimal discomfort to the patient. The damaged tissues should be easily fixed with the desired functions quickly restored. Another important factor controlling the designing of tissue culture is the source of donor cells. The cells from the patient himself, is always preferred as it considerably reduces the immunological complications. However under certain situations allogeneic cells (cells taken from a person other than the patient) are also used. The other important factors are the support material, its degradation products, cell adhesion characteristics etc. It was demonstrated in 1975 that human keratinocytes could be grown in the laboratory in a form suitable for grafting. A continuous sheet of epithelial cells can be grown now however there is still difficult to grow TE skin with the dermal layer with all the blood capillaries, nerves, sweat glands, and other accessory organs.Some of the implantable skin substitutes which are tissue engineering skin constructs with a limited shelf life of about 5 days are:a) Integra TM A bioartificial material composed of collagen-glycosaminoglycan and is mainly used to carry the seeded cells.b) DermagraftTM- This is composed of poly glycolic acid polymer mesh seeded with human dermal fibroblasts from neonatal foreskins.c) ApligrafTM- It is constructed by seeding human dermal fibroblasts into collagen gel with the placement of a layer of human keratinocytes on the upper surface.These tissue constructs integrate into the surrounding normal tissue and form a good skin cover with minimum immunological complications.The urothelial cells and smooth muscle cells from bladder are now being cultured and attempts are on to construct TE urothelium. Some progress has also been made in the repair of injured peripheral nerves using tissue engineered peripheral nerve implants. The regeneration of the injured nerve occurs from the proximal stump to rejoin at distal stump.The regeneration process requires substances like-

(a) Conduct material- The conduct material is composed of collagen- glycosaminoglycans, PLGA (poly lactic- co- glycolicacid), hyaluronan and fibronectin and forms the outer layer.

(b) Filling material- The filling material contains collagen, fibrin, fibronectin and agarose. This supports the neural cells for regeneration. and

(c) Additives- A large number of other factors are also added e.g. growth factors, neurotrophic factors such as fibroblast growth factor (FGF), nerve growth factor (NGF).The other important applications of tissue engineering are in gene therapy, pseudo-organs and as model cell systems for developing new therapeutic approaches to human diseases.The attempts are on to create tissue models in the form of artificial organs using tissue engineering. The artificial liver is being created using hepatocytes cultured as spheroids and held suspended in artificial support system such as porous gelatin sponges, agarose or collagen. Some progress has been made in the area of creating the artificial pancreas using spheroids of insulin secreting cells which have been developed from mouse insulinoma beta cells.Three dimensional brain cell cultures have been used for the study of neural myelination, neuronal regeneration, and neurotoxicity of lead. The aggregated brain cells are also being used to study Alzheimers disease and Parkinsons disease. Thyroid cell spheroids are being used to study cell adhesion, motility, and thyroid follicle biogenesis. (Table 8.2 page 155, gupta)TABLE DEPICTING THE TECHNOLOGICAL GOALS AND AREAS OF RESEARCH IN TISSUE ENGINEERINGGrowth of cells in three- dimensional systems

Delivery systems for protein therapeutics

Cell cultivation methods for culturing recalcitrant cells

Expression of transgenic proteins in transplantable cells

To develop vehicles for delivering transplantable cells

Development of markers for tracking transplanted cells

Avoiding immunogenicity in transplantable cells

Development of in vivo and ex vivo biosensors for monitoring cellbehaviour during tissue production

Valuable cell culture products :

Insulin: The complex differentiation pattern of hES cells includes a subset of cells that have many characteristics of -cell function, including proinsulin and/or insulin production and insulin release, as well as the expression of other -cell markers. Although not surprising, in view of the capacity of ES cells to differentiate into multiple different cell types, this finding is a necessary prerequisite for therapeutic strategies based on cell enrichment from hES cells as a source of cell replacement in type 1 diabetes.

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 VIIIHaemophilia 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.

Erythropoietin (EPO)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 technologyAntibodies 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 calledabzymes.Using animal cell cultures, it is also possible to producePolyclonal Antibodies. Polyclonal antisera are derived from many cells therefore contains heterogeneous antibodies that are specific for several epitopes or an antigen.