Download Growth of Animal Cells in Culture

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
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.
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 501–504
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,
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.
Figure 1.40. Culture of animal cells
©2000 by Geoffrey M. Cooper