Cell Culture Basics | 27
Part 4. Methods
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 Density
• Mammalian cells: Adherent 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.
• Insect cells: Insect 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 decreased viabilities,
and are considered unhealthy.
Exhaustion of Medium
• Mammalian cells: A drop in the pH of the growth medium usually indicates a build up
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 cells: 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
Passaging your cells according 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.