Cells are grown and maintained at an appropriate temperature and gas mixture
Cells
are grown and maintained at an appropriate temperature and gas mixture
(typically, 37 °C, 5% CO2 for mammalian cells) in a cell incubator.
Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes.
Aside from temperature
and gas mixture, the most commonly varied factor in culture systems is
the cell growth medium. Recipes for growth media can vary in pH, glucose
concentration, growth factors, and the presence of other nutrients. The
growth factors used to supplement media are often derived from animal
blood, such as calf serum. One complication of these blood-derived
ingredients is the potential for contamination of the culture with
viruses or prions, particularly in medical biotechnology applications.
Current practice is to minimize or eliminate the use of these
ingredients wherever possible and use chemically defined media, but this
cannot always be accomplished. Alternative strategies involve sourcing
the animal blood from countries with minimum BSE/TSE risk, such as
Australia and New Zealand, and using purified nutrient concentrates
derived from serum in place of whole animal serum for cell culture.[5]
Also the use of recently developed universal, fully defined and animal
free alternatives like Serum-Free avoids these complications.culture medium
Plating density (number
of cells per volume of culture medium) plays a critical role for some
cell types. For example, a lower plating density makes granulosa cells
exhibit estrogen production, while a higher plating density makes them
appear as progesterone-producing theca lutein cells.
Cells can be grown either in suspension or adherent cultures. Some
cells naturally live in suspension, without being attached to a surface,
such as cells that exist in the bloodstream. There are also cell lines
that have been modified to be able to survive in suspension cultures so
they can be grown to a higher density than adherent conditions would
allow. Adherent cells require a surface, such as tissue culture plastic
or microcarrier, which may be coated with extracellular matrix
components to increase adhesion properties and provide other signals
needed for growth and differentiation. Most cells derived from solid
tissues are adherent. Another type of adherent culture is organotypic
culture, which involves growing cells in a three-dimensional
(3-D) environment as opposed to two-dimensional culture dishes. This 3D
culture system is biochemically and physiologically more similar to in
vivo tissue, but is technically challenging to maintain because of many
factors (e.g. diffusion).cultural media
subculture or splitting cells
Pass aging
(also known as subculture or splitting cells) involves transferring a
small number of cells into a new vessel. Cells can be cultured for a
longer time if they are split regularly, as it avoids the senescence
associated with prolonged high cell density. Suspension cultures are
easily passaged with a small amount of culture containing a few cells
diluted in a larger volume of fresh media. For adherent cultures, cells
first need to be detached; this is commonly done with a mixture of
trypsin-EDTA; however, other enzyme mixes are now available for this
purpose. A small number of detached cells can then be used to seed a new
culture.
manipulating cells involves the introduction of foreign DNA by transfection.
Another common method for manipulating cells involves the introduction of foreign DNA
by transfection. This is often performed to cause cells to express a
protein of interest. More recently, the transfection of RNAi constructs
have been realized as a convenient mechanism for suppressing the
expression of a particular gene/protein. DNA
can also be inserted into cells using viruses, in methods referred to
as transduction, infection or transformation. Viruses, as parasitic
agents, are well suited to introducing DNA into cells, as this is a part
of their normal course of reproduction
hybridoma of proliferation
It is possible to fuse normal cells with an immortalised cell line. This method is used to produce monoclonal
antibodies. In brief, lymphocytes isolated from the spleen (or possibly
blood) of an immunised animal are combined with an immortal myeloma
cell line (B cell lineage) to produce a hybridoma which has the antibody
specificity of the primary lymphoctye and the immortality of the
myeloma. Selective growth medium (HA or HAT) is used to select against
unfused myeloma cells; primary lymphoctyes die quickly in culture and
only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.
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