Growth of Animal Cells in Culture

Transcription

1 Growth of Animal Cells in Culture The ability to study cells depends largely on how readily they can be grown and manipulated in the laboratory. Although the process is technically far more difficult than the culture of bacteria or yeasts, a wide variety of animal and plant cells can be grown and manipulated in culture. Such in vitro cell culture systems have enabled scientists to study cell growth and differentiation, as well as to perform genetic manipulations required to understand gene structure and function. Animal cell cultures are initiated by the dispersion of a piece of tissue into a suspension of its component cells, which is then added to a culture dish containing nutrient media. Most animal cell types, such as fibroblasts and epithelial cells, attach and grow on the plastic surface of dishes used for cell culture. Because they contain rapidly growing cells, embryos or tumors are frequently used as starting material. Embryo fibroblasts grow particularly well in culture and consequently are one of the most widely studied types of animal cells. Under appropriate conditions, however, some specialized cell types can also be grown in culture, allowing their differentiated properties to be studied in a controlled experimental environment. The culture media required for the propagation of animal cells are much more complex than the minimal media sufficient to support the growth of bacteria and yeasts. Early studies of cell culture utilized media consisting of undefined components, such as plasma, serum, and embryo extracts. A major advance was thus made in 1955, when Harry Eagle described the first defined media that supported the growth of animal cells. In addition to salts and glucose, the media used for animal cell cultures contain various amino acids and vitamins, which the cells

2 cannot make for themselves. The growth media for most animal cells in culture also include serum, which serves as a source of polypeptide growth factors that are required to stimulate cell division. Several such growth factors have been identified. They serve as critical regulators of cell growth and differentiation in multicellular organisms, providing signals by which different cells communicate with each other. For example, an important function of skin fibroblasts in the intact animal is to proliferate when needed to repair damage resulting from a cut or wound. Their division is triggered by a growth factor released from platelets during blood clotting, thereby stimulating proliferation of fibroblasts in the neighborhood of the damaged tissue. The identification of individual growth factors has made possible the culture of a variety of cells in serum-free media (media in which serum has been replaced by the specific growth factors required for proliferation of the cells in question). The initial cell cultures established from a tissue are called primary cultures (Figure 1.40). The cells in a primary culture usually grow until they cover the culture dish surface. They can then be removed from the dish and replated at a lower density to form secondary cultures. This process can be repeated many times, but most normal cells cannot be grown in culture indefinitely. For example, normal human fibroblasts can usually be cultured for 50 to 100 population doublings, after which they stop growing and die. In contrast, cells derived from tumors frequently proliferate indefinitely in culture and are referred to as immortal cell lines. In addition, a number of immortalized rodent cell lines have been isolated from cultures of normal fibroblasts. Instead of dying as most of their counterparts do, a few cells in these cultures continue proliferating indefinitely, forming cell lines like those derived from tumors. Such permanent cell lines have been particularly useful for many types of experiments because they provide a continuous and uniform source of cells that can be manipulated, cloned, and indefinitely propagated in the laboratory.

3 Even under optimal conditions, the division time of most actively growing animal cells is on the order of 20 hours ten times longer than the division time of yeasts. Consequently, experiments with cultured animal cells are more difficult and take much longer than those with bacteria or yeasts. For example, the growth of a visible colony of animal cells from a single cell takes a week or more, whereas colonies of E. coli or yeast develop from single cells overnight. Nonetheless, genetic manipulations of animal cells in culture have been indispensable to our understanding of cell structure and function. Key Experiment : Animal Cell Culture Nutrition Needs of Mammalian Cells in Tissue Culture Harry Eagle National Institutes of Health, Bethesda, MD Science, Volume 122, 1955, pages The Context The earliest cell cultures involved the growth of cells from fragments of tissue that were embedded in clots of plasma a culture system that was far from suitable for experimental analysis. In the late 1940s, a major advance was the establishment of cell lines that grew from isolated cells attached to the surface of culture dishes. But these cells were still grown in undefined media consisting of varying combinations of serum and embryo extracts. For example, a widely used human cancer cell line (called HeLa cells) was initially established in 1952 by growth in a medium consisting of chicken plasma, bovine embryo extract, and human placental cord serum. The use of such complex and undefined culture media made analysis of the specific growth requirements of animal cells impossible. Harry Eagle was the first to solve this problem,

4 by carrying out a systematic analysis of the nutrients needed to support the growth of animal cells in culture. The Experiments Eagle studied the growth of two established cell lines: HeLa cells and a mouse fibroblast line called L cells. He was able to grow these cells in a medium consisting of a mixture of salts, carbohydrates, amino acids, and vitamins, supplemented with serum protein. By systematically varying the components of this medium, Eagle was able to determine the specific nutrients required for cell growth. In addition to salts and glucose, these nutrients included 13 amino acids and several vitamins. A small amount of serum protein was also required. The basal medium developed by Eagle is described in the accompanying table, reprinted from his 1955 paper. The Impact The medium developed by Eagle is still the basic medium used for animal cell culture. Its use has enabled scientists to grow a wide variety of cells under defined experimental conditions, which has been critical to studies of animal cell growth and differentiation, including identification of the growth factors present in serum now known to include polypeptides that control the behavior of individual cells within intact animals.

5 Figure Culture of animal cells 2000 by Geoffrey M. Cooper

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