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Historical Development of Animal Cell Culture

The history of animal cell culture began about a century ago. In 1907, Ross Harrison made the first attempt to grow the animal cells in culture.

Using hanging drop technique, he was able to cultivate the embryonic nerve cells of a frog in a blood clot. Barrows and others extended this work and developed a technique for cultivation of a variety of mammalian cells.

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Alexis Carrel (1912) used tissue and embryo extract as culture media. After using the mixture of chick embryo extract and plasma, cell proliferation was enhanced in vitro. The fibrin clot of plasma served as an anchor for cell attachment and the extract provided growth factors and nutrients.

A major innovation to get cell suspension was the use of trypsin for cell disaggregation from tissue explants. This allowed single cell culture. Thus the technique of cell cultures differs from tissue culture. The use of biological fluids and extracts was a major problem because of getting contaminated.

During the late 1940s, the discovery of antibiotics led to the development of improved cell culture techniques. Such studies provided an impetus for culture of animal cells in large scale.

During this period many human carcinoma cell lines (e.g. HeLa cell line) were isolated and grown in culture.

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Alexis Carrel, the famous physiologist kept the chick embryo heart alive and its beating continued in vitro for about three months. Animal cell culture studies resulted in total stop .of using monkeys for multiplication of animal viruses.

In the late 1940s, Enders, Weller and Robbins grew poliomyelitis virus in culture. They HeLja cell culture, provided an impetus to give an easy way to test many chemicals and antibiotics that affect multiplication of virus in living host cells.

In 1950s, animal cell culture technology began to produce polio vaccine. Development of first synthetic culture medium by Earle and co-workers was a milestone in animal cell culture technology.

The synthetic media contain all the vitamins, amino acids and minerals. Foetal calf serum supports cell growth in vitro.

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Serum contains a complex mixture of proteins, hormones and other growth factors. At present several media have been developed for animal cell and tissue culture. Tissue culture method provided the animal virologists a tool for in vitro propagation of many viruses.

In 1966, Alec Issacs infected the cultured cells with viruses and collected filtrate from the infected cells. Again he cultured fresh cells in medium containing filtrate.

Such cells were not infected when challenged with viruses. He predicted that the virus-infected cells secreted molecules which coated the surface of fresh cells (uninfected cells).

These molecules interfered the entry of viral particles in the uninfected cells. Issac called these molecules as interferon. This interpretation could not easily be accepted by the scientific community. It was also said that Issac was mad.

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In 1980, the validity of Issac’s observation was proved through recombinant DNA technology by cloning and expression of interferon gene in bacterial cells. Now interferon a, (i and y are one of the most successful biotech products which are available in market.

Chinese Hamster ovary (CHO) cell lines were developed during 1980s. Recombinant erythropoietin was produced on CHO cell lines by AMGEN (U.S.A.). Erythropoietin stimulated RBC formation.

It is used to prevent anaemia in patients with kidney failure who require dialysis. Within 10 years AMGEN became the first billion biotech company only due to erythropoietin.

The Food and Drug Administration (U.S.A.) granted the approval for manufacturing erythropoietin on CHO cell lines (see the preceding section and Table 9.1). Attempts are being made to culture animal cells for commercial production of recombinant products of animals as the microbial and plant cells are used.

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The mammalian cells can be grown in industrial scale cultures in multiple low-productivity roller bottles. In 1982, Thilly and co-workers grew certain mammalian cells to densities as high as 5xl06 cells/ml by employing the conventional conditions of medium, serum and O2 and using Fig. 9.1: Coral grafts – as a substitute suitable beads as carriers, for bone.

A tremendous excitement has been done in the area of medical science with the developraenl of stem cell technology. It holds a great potential to replace the damaged and dead cells (of blood cancer), neuro-degenerative diseases, etc.

Since such cells are used as therapeutic cells, hence it is also called cell-based therapy. Attempts are also made in the similar direction for in vitro culture of cardiac tissue, neuronal tissue, blood capillaries, etc.

The untiring efforts have increased the scientific ethos to fulfil the dream of Biocivil engineering (i.e. replacement of defective organs by in vitro grown organs).

If manifestation of such imagination becomes the reality, the organ transplantation (such as heart, kidney, liver, etc.) in patients would be an easier game for the scientists and a boon to the patients.

In February 1997, Wilmut and co-workers announced that they have successfully produced a transgenic sheep named Dolly through nuclear transfer technique. But Dolly was born in July 1996.

Dolly was cloned by transferring nucleus of a mammary (udder) cell into enucleated ovum of an adult sheep. Thereafter, many such animals (like sheep, goat, pigs, fishes, birds, etc.) were produced.

In 1999 was speculated that Dolly may have been susceptible to premature aging, due to shortened telomere in her cells.

Because these were passed on from her parent, who was six years old when the genetic material was taken from her, so that Dolly may have been genetically six years old at birth. Moreover, Dolly gave birth to a baby, called Bonnie.

After one year (officially after five months) of birth of Dolly, on 24 July 1997, PPL Therapeutics announced that a ‘genetically engineered’ lamb called Polly had been produced by the same method of nuclear transfer that had produced Dolly.

In addition to her usual complement of sheep genes, Polly also contained a human gene which had been added to the cells while they were still a cell culture. The purpose for introduction of a gene was to produce a therapeutically useful protein in the milk of the sheep.

In January 2002, when Dolly was five years old, possible signs of her condition were reported. She had developed a potentially debilitating form of arthritis at an unusually early age. This supported the theory of premature senescence. Dolly died on 14th February, 2003 due to a progressive lung disease.

An autopsy confirmed she had ‘ovine pulmonary adenocarcinoma, which is a fairly common disease of sheep. Scientists stated that they did not think there was a connection with Dolly being a clone.

Other sheep on the farm had similar ailments. Such lung diseases are especially a danger for sheep kept indoors, as Dolly was kept for security reasons.

In 2002, Clonaid, a Human Genome Society of France claimed to produce a cloned human baby named Eve.

The Government of India is promoting cloning of cattle and other animals. For this endeavour research work has been started in certain laboratories of the Department of Biotechnology, Government of India.

The National Institute of Immunology (New Delhi) has prepared genetically engineered more than 17 mice for various experiments in the area of healthcare and genetic improvement in livestock.

Similarly Centre for Cellular and Molecular Biology (CCMB) (Hyderabad) has developed transgenic fly by transferring cancer causing genes. Similarly National Dairy Research Institute (Karnal) has developed transgenic cows which can give more milk rich in certain proteins.

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