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The QIAGEN Guide to Animal Cell Culture

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 be discarded.

Tip - The Transfection Tools web site ( 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

Incubation conditions

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 nt of animal cells to the growing surface are available commercially.

Cell banking

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.

Culture instability

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 Interscience.



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