Guidelines to Maintain Cultured Cells

The growth of cells in culture follows a standard pattern. A lag after seeding is followed by a period of exponential growth, called the log phase.  When the cells in adherent cultures occupy all the available substrate and have no room left for expansion, or when the cells in suspension cultures exceed the capacity of the medium to support further growth, cell proliferation is greatly reduced or ceases entirely (see Figure 4.1 below). Cells should be passaged, or subcultured, when they cover the plate, or the cell density exceeds the capacity of the medium. This will keep cells at an optimal density for continued growth and will stimulate further proliferation. Maintaining log phase growth will maximize the number of healthy cells for your experiment.

Figure 4.1:  Characteristic Growth Pattern of Cultured Cells.  The semi-logarithmic plot shows the cell density versus the time spent in culture.  Cells in culture usually proliferate following a standard growth pattern.  The first phase of growth after the culture is seeded is the lag phase, which is a period of slow growth when the cells are adapting to the culture environment and preparing for fast growth.  The lag phase is followed by the log phase (i.e., “logarithmic” phase), a period where the cells proliferate exponentially and consume the nutrients in the growth medium.  When all the growth medium is spent (i.e., one or more of the nutrients is depleted) or when the cells occupy all of the available substrate, the cells enter the stationary phase (i.e., plateau phase), where the proliferation is greatly reduced or ceases entirely.

The criteria for determining the need for subculture are similar in adherent and suspension cultures; however, there are some differences between mammalian and insect cell lines.

When conducting cell passaging, adhering to a strict schedule ensures reproducible behavior and allows you to monitor their health status.  Vary the seeding density of your cultures until you achieve consistent growth rate and yield appropriate for your cell type from a given seeding density.  Deviations from the growth patterns thus established usually indicate that the culture is unhealthy (e.g., deterioration, contamination) or a component of your culture system is not functioning properly (e.g., temperature is not optimal, culture medium too old).  We strongly recommend that you keep a detailed cell culture log, listing the feeding and subculture schedules, types of media used, the dissociation procedure followed, split ratios, morphological observations, seeding concentrations, yields, and any anti-biotic use.

It is best to perform experiments and other non-routine procedures (e.g., changing type of media) according to your subculture schedule.  If your experimental schedule does not fit the routine subculture schedule, make sure that you do not passage your cells while they are still in the lag period or when they have reached confluency and ceased growing.

Many continuous mammalian cell lines can be maintained on a relatively simple medium such as MEM supplemented with serum, and a culture grown in MEM can probably be just as easily grown in DMEM or Medium 199.  However, when a specialized function is expressed, a more complex medium may be required.   Information for selecting the appropriate medium for a given cell type is usually available in published literature and may also be obtained from the source of the cells or cell banks.

If there is no information available on the appropriate medium for your cell type, choose the growth medium and serum empirically or test several different media for best results.  In general, a good place to start is MEM for adherent cells and RPMI-1640 for suspension cells.

Insect cells are cultured in growth media that are usually more acidic than those used for mammalian cells such as TNM-FH and Grace’s medium.

• Recommended Media for Common Cell Types

The first step in subculturing adherent cells is to detach them from the surface of the culture vessel by enzymatic or mechanical means.  The table below lists the various cell dissociation procedures.

Gibco TrypLE Express and TrypLE Select are microbially produced cell dissociation enzymes with similar kinetics and cleavage specificities to trypsin.  Although Gibco™ TrypLE™ enzymes can directly substitute for trypsin in dissociation procedures without a need for protocol changes, we recommend that you initially optimize the incubation time for dissociation.  Because TrypLE enzymes are recombinant fungal trypsin-like proteases, they are ideal for applications that require animal origin-free reagents.  The table below compares TrypLE Express and TrypLE Select to trypsin.