Part III: Cell culture conditions
Welcome to the third of a series of articles providing useful hints for
culturing animal cells. This article describes general considerations for
cell culture conditions. The series will continue in future issues of QIAGEN
News with cell culture protocols, followed by hints for successful transfection.
Media and serum
The choice of cell culture medium is extremely important, and significantly
affects the success of cell culture experiments. Different cell types have
highly specific growth requirements, and the most suitable medium for each
cell type must be determined experimentally. Common basal media include
Eagle minimal essential medium (MEM), Dulbeccos modified Eagle medium
(DMEM), RPMI 1640, and Ham F10. These contain a mixture of amino acids,
glucose, salts, vitamins, and other nutrients, and are available either
as a powder or as a liquid from various commercial suppliers.
Basal media are usually supplemented just before use with serum, L-glutamine,
and antibiotics and/or fungicides to give complete medium (also called growth
medium). Serum is a partially undefined material that contains growth and
attachment factors, and may show considerable variation in the ability to
support growth of particular cells. Fetal calf serum (FCS) is the most frequenty
used serum, but for some applications less expensive sera such as horse
or calf serum can be used. Different serum batches should be tested to find
the best one for each cell type. L-glutamine is an unstable amino acid that,
with time, converts to a form that cannot be used by cells, and should be
added to medium just before use. Antibiotics and
fungicides can be used
as a supplement to aseptic technique to prevent microbial contamination.
The working concentration of commonly used antibiotics and fungicides is
provided in Table 1. Some cell types, particularly primary cells, require
additional supplements (e.g., collagen and fibronectin, hormones such as
estrogen, and growth factors such as epidermal growth factor and nerve growth
factor) to attach to the cell culture vessel and proliferate.
Tip - Media, serum, and supplements should be tested for sterility before
use by incubation of a small aliquot at 37C for 48 hours. If microbial
growth has occurred after this incubation, the medium or supplement should
Tip - The Transfection Tools web site (www.qiagen.com/transfectiontools/)
has an online cell database that includes information about media used with
different cell types. Click on the cell type of interest, and then click
on Detail to view this information.
Table 1. Commonly used antibiotics and fungicides for animal cell culture
The incubation conditions used to culture cells are also important. Cell
cultures should be incubated in an incubator with a tightly regulated temperature
(e.g., a water-jacketed incubator) and CO2 concentration. Most cell lines
grow at 37C and 5% CO2 with saturating humidity, but some cell types
require incubation at lower temperatures and/or lower CO2 concentrations.
Cell culture vessel
The choice of growth vessel can influence the growth of adherent cells.
Sterile, disposable dishes and flasks that have been treated to allow attachme
of animal cells to the growing surface are available commercially.
For some cell cultures, especially those that are valuable, it is common
practice to maintain a two-tiered frozen cell bank: a master cell bank and
a working cell bank. The working cell bank comprises cells from one of the
master bank samples, which have been grown for several passages before storage.
If future cell samples are needed, they are taken from the working cell
bank. The master cell bank is used only when absolutely necessary. This
ensures that a stock of
cells with a low passage number is maintained, and avoids genetic variation
within the cell culture.
The growth rate of cells that have been repeatedly subcultured may sometimes
unexpectedly decrease, and the cytotoxicity of, for example, a transfection
process may unexpectedly increase. This instability can result from variations
in cell culture conditions, genomic variation, and selective overgrowth
of constituents of the cell population. We recommend using cells with a
low passage number (<50 splitting cycles). To safeguard against instability
in continuous cell lines, avoid senescence or transformation in finite cell
lines, and maintain consistency in transfection experiments, we recommend
creating cell banks by freezing aliquots of cells to recall into culture
if and when necessary.
1. Ausubel, F.M. et al. eds.
(1991) Current protocols
in molecular biology.
New York: Wiley
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