Cell culture conditions

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), Dulbecco’s 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 frequently 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 the tables Commonly used antibiotics for animal cell culture and Commonly used fungicides for animal cell culture. 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.

Media, serum, and supplements should be tested for sterility before use by incubation of a small aliquot at 37°C for 48 hours. If microbial growth has occurred after this incubation, the medium or supplement should be discarded.

Commonly used antibiotics for animal cell culture

Antibiotic	Working concentration	Effective against	Stability at 37°C
Penicillin	50–100 U/ml	Gram-positive bacteria	3 days
Streptomycin	50–100 µg/ml	Gram-negative bacteria	5 days
Kanamycin	100 µg/ml	Gram-positive and gram-negative bacteria; mycoplasma	5 days
Gentamycin	5–50 µg/ml	Gram-positive and gram-negative bacteria; mycoplasma	5 days

Commonly used fungicides for animal cell culture

Antibiotic	Working concentration	Effective against	Stability at 37°C
Nystatin	100 U/ml	Yeasts and molds	3 days
Amphotericin B	0.25–2.5 µg/ml	Yeasts and molds	3 days

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 37°C 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 attachment 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 (<10 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.