appropriate seeding density. If cells are seeded at a lower density they may take longer
to reach confluency and some may die before getting to this point. On the other hand,
if seeded at too high a density cells will reach confluency too quickly, resulting in
irreproducible experimental results as already discussed above (see Section 2.5.6).
The seeding density will vary depending on the cell type and on the surface area of
the culture flask into which the cells will be placed. These factors should therefore
be taken into account when deciding on the seeding density of any given cell type
and the purpose of the experiments carried out.2.5.8 Maintenance of cells in culture
It is important that after seeding, flasks are clearly labelled with the date, cell type and
the number of times the cells have been subcultured or passaged. Moreover, a strict
regime of feeding and subculturing should be established that permits cells to be fed
at regular intervals without allowing the medium to be depleted of nutrients or the
cells to overgrow or become super confluent. This can be achieved by following a
standard but routine procedure for maintaining cells in a viable state under optimum
growth conditions. In addition, cultures should be examined daily under an inverted
microscope, looking particularly for changes in morphology and cell density. Cell
shape can be an important guide when determining the status of growing cultures.
Round or floating cells in subconfluent cultures are not usually a good sign and may
indicate distressed or dying cells. The presence of abnormally large cells can also
be useful in determining the well-being of the cells, since the number of such cells
increases as a culture ages or becomes less viable. Extremes in pH should be avoided
by regularly replacing spent medium with fresh medium. This may be carried out on
alternate days until the cultures are approximately 90% confluent, at which point the
cells are either used for experimentation or trypsinised and subcultured following the
procedures outlined in Section 2.5.5.
The volume of medium added to the cultures will depend on the confluency of the
cells and the surface area of the flasks in which the cells are grown. As a guide, cells
which are under 25% confluent may be cultured in approximately 1 cm^3 of medium
per 5 cm^2 and those between 25% and 40% or≧45% confluency should be supple-
mented with 1.5 cm^3 or 2 cm^3 culture medium per 5 cm^2 , respectively. When changing
the medium it is advisable to pipette the latter on to the sides or the opposite surface of
the flask from where the cells are attached. This is to avoid making direct contact with
the monolayers as this will damage or dislodge the cells.2.5.9 Growth kinetics of animal cells in culture
When maintained under optimum culture conditions, cells follow a characteristic
growth pattern (Fig. 2.8), exhibiting an initiallag phasein which there is enhanced
cellular activity but no apparent increase in cell growth. The duration of this phase is
dependent on several factors including the viability of the cells, the density at which
the cells are plated and the media component.58 Cell culture techniques