Personal Study: Virus Culture
Unlike most bacteria that can be grown in artificial media (eg agar
plates, nutrient broth) viruses cannot be grown on artificial media
but must be grown in living cells.
There are only three ways in which viruses can be grown in the
- In a living host such as an animal or plant (if it is a plant
- In an embryonated egg. The virus grows in the developing chick
embryo or associated tissues
- In tissue or cell culture. Most commonly today cells derived
from tissues rather than the tissues themselves are used. Cell
culture has many advantages, such as:
- no use of whole animals
- cells keep growing and are a renewable resource
- many different kinds of cells can be used, including human cells
- cells can be grown in various containers and numbers to suit
- surplus cells can be stored indefinitely in liquid nitrogen
Types of cell cultures
|Primary Cell Culture
||Diploid Cell Lines (or strains)
||Continuous Cell Lines
|Developed directly from living tissue and contain several
different kinds of cells. They are expensive as they cannot
be subcultured (passaged) more than a few times and hence new
tissue needs to be obtained from animals on a regular basis.
One example of a primary cell line is chicken embryo fibroblasts.
||These cells can be subcultured about 100 times before they
die. Much more useful in the laboratory and they can be stored
indefinitely in liquid nitrogen. An example of this type is
human embryonic fibroblasts.
|These cells have lost the normal constraints on cell growth
such as contact inhibition and mortality. They can be subcultured
indefinitely in vitro. Examples include Hep-2, HeLa (from
human cancers) and VERO (from Green African monkey kidney).
Growth of cell lines
Cells are grown under stringent aseptic conditions, as the media
that the cells are grown in is rich in a wide range of nutrients
and will support the growth of a wide range of opportunistic bacterial
contaminants. Mycoplasma sp. are a particular nuisance as
they are hard to detect, are small and will pass through many filters,
and are found widely in the environment including in plants and
Cell culture media often includes a range of salts, vitamins, hormones
and other growth factors, amino acids or proteins, glucose, antimicrobial
agents, a buffering system, a pH indicator, and non-specific sources
of nutrients such as foetal bovine serum. Examples of cell culture
media include Medium M199, Eagle's Basal Medium and RPMI 1640.
Cells growing in cell culture attach to the surface of the container
(glass or specialised plastic) then start to divide and grow across
the surface. They form a sheet of cells one cell thick (monolayer)
and when the surface is covered it is called confluent. Many cells
stop growing once confluent (contact inhibition).
Some cells do not attach to surfaces and are grown in a suspension
culture, whilst cancer cells often keep growing and pile up forming
tiny heaps of cells.
Cells may be subcultured (called passaging) by:
- stripping the cells from the surface using trypsin, EDTA and
some gentle mechanical motion
- dispersing the cells in a suitable medium (often a 1:10 dilution
- finally seeding the cells into ten more containers or a container
ten times the size of the original
In this way significantly large numbers of cells can be grown.
Cells are incubated at an appropriate temperature (37°C for human
and mammalian cells, 28°C for insect cells) usually in an atmosphere
enriched in CO2 (to provide buffering with NaHCO3 in the cell culture
media) and humidified (to prevent cell cultures from drying out).
Growth of viruses in cell culture
Appropriate cell lines are infected with sterile preparations of
virus or suspected isolate (sterile means that all bacteria and
fungi have been excluded) and the cell line is incubated for a number
of days to allow the virus to grow in the infected cells. Remember
that the virus must grow in a living cell.
The cell culture is then inspected under a low magnification inverted
microscope and changes in the cells due to virus replication (cytopathic
effect or CPE) are looked for. Depending on the virus and cell line
used CPE can include rounding up (shrivelling) of the cell, loss
of patches of cells or all the cells from the monolayer, inclusion
bodies (dark aggregated viral material in the cell), or formation
of large fused areas of cells (syncitia).
Immunofluorescence can be used to detect the presence of specific
viral proteins in an infected cell culture using antibodies directed
against the viral protein and a Fluorescence microscope.
Viruses may also be harvested and purified from the cells of the
monolayer or the cell culture medium for further research or investigations.