Cell and Tissue Cultures

Cell Production and Tissue Cultures

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases.

However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas will overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammalian cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessary for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left alive.

A hybridoma can live indefinitely in a growth medium that includes salts, glucose, glutamine, certain amino acids, and bovine serum that provides essential components that have not been identified. Serum is expensive, and its cost largely determines the economic feasibility of a particular culture system. Only recently have substitutes or partial replacements for serum been found. Antibiotics are often included to prevent infection of the culture. The pH, temperature and dissolved oxygen, and carbon dioxide concentration must be closely controlled.

The salt determines the osmotic pressure to preserve the integrity of the fragile cell. Most glucose is metabolized to lactate because glycolysis is usually much faster than uptake rate of glycolytic intermediates. Glutamine acts as the primary source of nitrogen as well as providing additional carbon and energy. After glutamine is partially oxidized to glutamate, it can enter the TCA cycle and emerge as pyruvate. It has been estimated that between 30 and 65 percent of the cell energy requirement is derived from glutamine metabolism when both glucose and glutamine are available. Ammonia is produced in the deamination of glutamine to form glutamate and in the formation of alpha-ketoglutarate.

Plant Cells and Tissues It is estimated that today some 75 percent of all pharmaceuticals originate in plants. Typically, these compounds are derived from the secondary metabolic pathways of the cells. When plant or animal cells are cultured, concepts from microbiology come into play. Only specialized cells are used, and these can be improved with mutation, selection, and recombinant DNA techniques.

One very major difference between cell and tissue cultures and most microbiological processes is very high susceptibility to contamination by foreign organisms. Most microorganisms grow rapidly and compete well; some are aided by their own changes to the environment.

When a microbial process changes the pH to be far from neutrality or when the product such as ethanol is inhibitory to other organisms, growth of contaminants is discouraged. Cell and tissue cultures require rich media and are characterized by slow growth rates.

There is seldom any protection by the products of the process. Optimum conditions for production of the secondary metabolites are not likely to be the same as for growth. Economics may hinge on a good balance of growing sufficient cells and favoring product formation.

Only a few biochemicals derived from plant cell and tissue cultures have high volume/low value products, but some have sizeable markets as specialty chemicals such as dyes, fragrances, insecticides, and pesticides. These differ from the low volume/very high value compounds that typify life-saving drugs and pharmaceuticals. Examples for both of these categories are listed in Table 1 along with the plant species of origin.

Because of cell specialization, some products are produced in cultures of those cellular types. Three main classifications of the types of plant cell and tissue cultures are:

Undifferentiated cell cultures. Aggregate clumps of cells on solid media (callus) or in liquid media (suspension) Protoplast cultures. Cellular tissues devoid of cell wall material in culture Organ cultures. Differentiated tissues of shoots, roots, anthers, ovaries, or other plant organs in culture.