Tissue Culture and Storage
Tissue culture is defined as the propagation of plant and animal cells through the placement of small amounts of tissue in an artificial environment. Given the appropriate conditions, most kinds of plant and animal cells will live, even multiply and express differentiated properties in a tissue-culture dish.
Plant tissue culture was first used on a large scale by the orchid industry in the 1950s. Later, it became clear that any plant would respond to tissue culture as long as the right formula and the right processes were developed for its culture. In the case of plant tissue culture, a piece of plant, (which can be anything from a piece of stem, root, leaf, bud, or a single cell) is placed in a test tube. In a sterile environment (free from microorganisms) and in a balanced nutrient medium that bit of plant (explant) will form plantlets. These can multiply indefinitely if given proper care and later, can be taken out and planted normally. The medium for plant growth refers to a mixture of certain chemical compounds. By using tissue culture, one can control the environment and optimize it such that all the plants from the tissue culture are identical for the particular quality being sought, whether it be resistance to plant diseases or the production of a plant chemical.
Animal cell culture began in 1907, when a group of scientists decided to test the doctrine that states that each nerve fiber is the outgrowth of a single nerve cell and not a product of several cells. In order to test this, they placed small pieces of spinal cord on clotted tissue fluid in a warm moist chamber and observed it at regular intervals. After a day or so, individual nerve cells could be seen extending long thin processes into the clot. Thus the doctrine was validated and the foundation for cell-culture was laid.
Initially, small tissue fragments or explants were used for animal cell culture experiments. Today, however, cultures are more commonly made from suspensions of cells that have been dissociated from the tissues. These cells are propagated on a solid surface where they can grow and divide. Generally, a plastic culture dish is used to provide the mechanical support, and a nutrient medium is provided. Cells vary in their requirements and many will not grow unless their specific requirements are met. Liquid nutrient media containing well-defined mixtures of salts, amino acids, vitamins, etc are used for the propagation of cells. Most media also contain some proportion of a poorly defined biological material such as bovine serum, horse serum, or a crude extract from chick embryos. Since the specific growth and nutrient requirements cannot be determined using such media, various chemically defined media were developed. In these media, each component is a known molecule. In addition to these known molecules, chemically defined media also contain one or more of the various growth factors that most cells need to proliferate, such as "nerve growth factor" for nerve cells etc. The cells can be watched under the microscope and analyzed biochemically if necessary. The effects of adding or removing specific molecules such as hormones or growth factors can be investigated.
Cultures that are prepared directly from the tissues of an organism are called primary cultures. These primary cultures can be removed from the dish and seeded onto other dishes. This process known as "subculturing" can be used to form a large number of secondary cultures. They have to be repeatedly subcultured in this manner for weeks or even months. Cells cultured in this fashion often display the differentiated properties specific for their cell type, for e.g. fibroblasts will secrete collagen; nerve cells will extend axons and make synapses with other nerve cells; epithelial cells will form extensive sheets and so on.
Most vertebrate cells die after a finite number of divisions in culture, for e.g. human skin cells will typically last only several months in culture, dividing about 50-100 times. The limited life span is related to the limited life span of the animal from which they are derived. Occasionally, mutant cells (variants) arise that can be propagated indefinitely. These are known as "immortal cells". They are propagated as "cell lines" and grow best when attached to solid surfaces. They normally cease growing after they have formed a confluent layer over the surface of the culture dish.
Cell lines that are prepared from cancer cells differ from normal cell lines in that they grow without attaching to any cell surface and they proliferate to a very high density. Such cell lines are called "transformed cell lines." Normal cell lines can be "transformed" by inducing them with certain chemicals or a tumor-inducing virus. Both transformed and untransformed cell lines are extremely useful in cell research. They can be stored at -158°F (-70°C) for an indefinite period and are still viable when thawed.
In tissue culture, it is even possible to fuse one cell with another to form a combined cell with two separate nuclei called a "heterocaryon". To do this, the cells are treated with certain chemicals or viruses which alters the plasma membrane of the cells and induces them to fuse. Such cells are useful for studying the interactions between the components of two different cells and they also provide a convenient method for assigning genes to human chromosomes.
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