What is subculture?

Subculturing, also referred to as passaging cells, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.

Characteristic growth pattern of culture 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 showing characteristic growth patter for cultured cells
 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.

When to subculture?

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.

Cell LinesCell densityExhaustion of medium
Mammalian cellsAdherent cultures should be passaged when they are in the log phase, before they reach confluence.  Normal cells stop growing when they reach confluence (contact inhibition), and it takes them longer to recover when reseeded.  Transformed cells can continue proliferating even after they reach confluence, but they usually deteriorate after about two doublings. Similarly, cells in suspension should be passaged when they are in log-phase growth before they reach confluency.  When they reach confluency, cells in suspension clump together and the medium appears turbid when the culture flask is swirled.A drop in the pH of the growth medium usually indicates a buildup of lactic acid, which is a by-product of cellular metabolism. Lactic acid can be toxic to the cells, and the decreased pH can be sub-optimal for cell growth. The rate of change of pH is generally dependent on the cell concentration in that cultures at a high cell concentration exhaust medium faster than cells lower concentrations.  You should subculture your cells if you observe a rapid drop in pH (>0.1 – 0.2 pH units) with an increase in cell concentration.
Insect cellsInsect cells should be subcultured when they are in the log phase, before they reach confluency.  While tightly adherent insect cells can be passaged at confluency, which allows for easier detachment from the culture vessel, insect cells that are repeatedly passaged at densities past confluency display decreased doubling times, decreased viabilities, and a decreased ability to attach.  On the other hand, passaging insect cells in adherent culture before they reach confluency requires more mechanical force to dislodge them from the monolayer. When repeatedly subcultured before confluency, these cells also display decreased doubling times and display decreased doubling times, decreased viabilities, and are considered unhealthy.Insect cells are cultured in growth media that are usually more acidic that those used for mammalian cells.  For example, TNM-FH and Grace’s medium used for culturing Sf9 cells has a pH of 6.2.  Unlike mammalian cell cultures, the pH rises gradually as the insect cells grow, but usually does not exceed pH 6.4.  However, as with mammalian cells, the pH of the growth medium will start falling when insect cells reach higher densities.

Subculture schedule

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.

Media recommendations for common cell lines

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.

Dissociating adherent cells

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.

Dissociation Agent
Gentle shaking or rocking of culture vessel, or vigorous pipetting.
Loosely adherent cells, mitotic cells
Cell scraper
Cell lines sensitive to proteases; may damage some cells
Enzymatic dissociation
Strongly adherent cells
Enzymatic dissociationTrypsin + Collagenase
High density cultures, cultures that have formed multiple layers, especially fibroblasts
Enzymatic dissociationDispase
Detaching epidermal cells as confluent, intact sheets from the surface of culture dishes without dissociating the cells
Enzymatic dissociationGibco™ TrypLE™ dissociation enzyme
Strongly adherent cells; direct substitute for trypsin; applications that require animal origin-free reagents

TrypLE dissociation enzymes

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.

TrypLE Express and TrypLE Select
Completely free of animal- and human derived components
Porcine- or bovine-derived
Stable at room temperature for at least six months
Not stable at room temperature
Does not require inactivation
Requires inactivation with serum or other inhibitors

Video resources

Passaging cells

This video explains why, when, and how to passage cells grown in both adherent and suspension cultures.

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Freezing cells

This video demonstrates the critical steps required to freeze cells while maintaining optimal cell health.

Related protocol:

Thawing cells

This video presents the best way to thaw cells without harming them in this stressful process.

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