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Personal Study: Virus Culture

Introduction
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 laboratory.

  • In a living host such as an animal or plant (if it is a plant virus)

  • 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 needs
    - surplus cells can be stored indefinitely in liquid nitrogen (-196°C).

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 animals.

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 is used)
  • 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.


 


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