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Cell culture

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Epithelial cells in culture, stained for keratin (red) and DNA (green)
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Epithelial cells in culture, stained for keratin (red) and DNA (green)

Cell culture is the process by which either prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a routine laboratory technique in the 1950s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th Century.

Contents

History

19th Century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.[link] In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture. Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907-1910, establishing the methodology of tissue culture. Schiff, Judith Ann. Yale Alumni Magazine, February 2002.

Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The Salk polio vaccine was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.

Concepts

Culture conditions (for example growth media, pH, temperature) vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. This article is concerned with modern culture methods. At present, this article is biased toward the culture of mammalian cells.

A cell line is grown and maintained in the favourable conditions (typically, 37°C, 5% CO2) in a cell incubator. Most cell lines, with the exception of some derived from tumours, originate from cell cultures with a limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability. An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene.

Sometimes, it is possible to fuse normal cells with an immortal cell line. An example is the way monoclonal antibodies are made: Lymphocytes isolated from the blood of an immunised animal are combined with hybridoma cell lines in a selective growth medium: only the fused cells survive.

Cell culture methods can be applied to either s, such as bacteria or eukarytotic microorganisms, or to cells removed from a multi-cellular tissue. Related to cell culture are tissue culture and organ culture, which refer to methods for growing pieces of tissue or entire organs removed from an organism in an artificial environment. "Tissue culture" has lost much of its original meaning in that it is now used generically for cell culture of mammalian cells or more broadly any cell type removed from a multicellular organism; however, when used without a qualifier it is usually interpreted as meaning mammalian cell culture. Ultimately, all cell culture applied to cells from multicellular organisms starts with the removal of cells from a tissue, which is likely where the blurring of the distinction between "tissue culture" and "cell culture" derives.

The culture of viruses requires the culture of cells as hosts for the growth and replication of the virus.

Cells can be cultured for a longer time if they are split regularly. Growth medium is then replaced and the cells are diluted (after first detaching them by trypsin of NaOH from the support). The key to success in culturing cells is to mimic the environment in which they found themselves before being transplanted to an artificial environment. This environment is often an extracellular fluid derived from blood. Ham's tissue culture medium, a commonly used medium for mammalian cells, is an attempt to recreate this extracellular fluid.

Some cells naturally live without attaching to a surface, such as cells that exist in the bloodstream. Others require a surface, such as most cells derived from solid tissues. Still others can live under either condition and exhibit different phenotypes depending on whether or not they are attached to a surface, such as yeast and many types of bacteria. Also, the substrate might or might not provide nutrients to the cells. In the case of most cells derived from tissues, nutrients are provided by a liquid broth that bathes cells attached to a surface. There are cells that require an "air-liquid interface" to grow properly; in this case the cells are often grown on a "raft" of organic material that floats on the surface of a nutrient broth and acts like a wet sponge, feeding the cells from underneath while their tops are exposed to the air. For bacteria and yeast, small quantities of cells are usually grown on a solid support that contains nutrients embedded in it, usually a gel such as agar, while large-scale cultures are grown with the cells suspended in a nutrient broth.

Many animal cell cultures require media containing ingredients derived from animal blood, such as calf serum. These blood-derived ingredients pose the potential for contamination of derived pharmaceutical products with animal viruses. Current practice is to minimize or eliminate the use of these ingredients where possible. Testing cell culture-derived products for the presence of viruses is useful, but does not provide absolute protection against the presence of unknown viruses.

Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the Supreme Court of California held in 1990 that human patients have no property rights in cell lines derived from organs removed with their consent. [ceb]

It is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be.(MacLeoud et al. 1999) The reason for this is that some cell lines exhibit vigorous growth and thus can cross-contaminate cultures of other cell lines, in time overgrowing and displacing the original cells. The most common contaminant is the HeLa cell line. While this may not be of significance when general properties such as cell metabolism are researched, it is highly relevant e.g. in medical research focusing on a specific type of cell. Results of such research will be at least flawed, if not outright wrong in their conclusion, with possible consequences if therapeutic approaches are developed based on it. (Masters 2002). See List of contaminated cell lines.

Applications

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biologicals produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified), must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin.

Vaccines

Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for flu vaccines is being funded by the United States government in areas as diverse as using the common cold as a vector and use of adjuvants. [cold as vector news] [cold as vector research abstract] [adjuvant]

Examples

Primate cell lines
Rat tumor cell lines
Mouse cell lines
Plant cell lines

See also

References and notes

  • MacLeod, R. A. F. et al. (1999): Widespread intraspecies cross-contamination of human tumour cell lines. International Journal of Cancer 83:555–563.
  • Masters, John R. (2002): HeLa cells 50 years on: the good,the bad and the ugly. Nature Reviews Cancer 2:315-319.

External links

 

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