Cell culture

Overview
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.

History
The 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. 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.

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 Prizefor their discovery of a method of growing the virus in monkey kidney cell cultures.

Isolation of cells
Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood, however only the white cellsare capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture.

Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have 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. There are numerous well established cell lines representative of particular cell types.

Maintaining cells in culture
Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrient components. The growth factors used to supplement media are often derived from animal blood, such as calf serum. These blood-derived ingredients pose the potential for contamination of derived pharmaceutical products with viruses or prions. Current practice is to minimize or eliminate the use of these ingredients where possible.

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. Cells grown unattached to a surface are referred to as suspension cultures. Other adherent cultures cells can be grown on tissue culture plastic, which may be coated with extracellular matrix components (e.g. collagen or fibronectin) to increase its adhesion properties and provide other signals needed for growth.

Manipulation of cultured cells
As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:


 * Nutrient depletion in the growth media
 * Accumulation of apoptotic/necrotic (dead) cells.
 * Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing known as contact inhibition.
 * Cell-to-cell contact can stimulate promiscuous and unwanted cellular differentiation.

These issues can be dealt with using tissue culture methods that rely on sterile technique. These methods aim to avoid contamination with bacteria or yeast that will compete with mammalian cells for nutrients and/or cause cell infection and cell death. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics can also be added to the growth media.

Amongst the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells.

Media changes
The purpose of media changes is to replenish nutrients and avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifugation and resuspended in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

Passaging cells
Passaging or splitting cells involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this was historically done with a mixture of trypsin-EDTA, however other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture.

Transfection and transduction
Another common method for manipulating cells involves the introduction of foreign DNA by transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realised as a convenient mechanism for suppressing the expression of a particular gene/protein.

DNA can also be inserted into cells using viruses, in methods referred to as transduction, infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction.

Established human cell lines
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.

It is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be. 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.

Generation of hybridomas
It is possible to fuse normal cells with an immortalised cell line. This method is used to produce monoclonal antibodies. In brief, 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.

Applications of cell culture
Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), andanticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified), currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants.

Tissue culture and engineering
Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo. I

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. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,  or the use of adjuvants.

Plant cell culture methods
Plant cell cultures are typically grown as cell suspension cultures in liquid medium or as callus cultures on solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin.

Bacterial/Yeast culture methods
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.

Viral culture methods
The culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts for the growth and replication of the virus. Wholewild type viruses, recombinant viruses or viral products may be generated in cell types other than their natural hosts under the right conditions. Depending on the species of the virus, infection and viral replication may result in host cell lysis and formation of a viral plaque.

Common cell lines

 * Human cell lines
 * National Cancer Institute's 60 cancer cell lines
 * MCF-7 (breast cancer)
 * MDA-MB-438 (breast cancer)
 * U87 (glioblastoma)
 * A172 (glioma)
 * HeLa (cervical cancer)
 * HL60 (promyelocytic leukemia)
 * A549 (lung cancer)
 * HEK 293 cells (kidney - original HEK line is contaminated with HeLa)
 * SHSY5Y Human neuroblastoma cells, cloned from a myeloma
 * Jurkat cell line, derived from a patient with T cell leukemia
 * BCP-1 cells (PEL)
 * Lncap (Prostate Cancer)


 * Primate cell lines
 * Vero (African green monkey Chlorocebus kidney epithelial cell line initiated 1962)
 * COS-7 (African Green Monkey Kidney Cells)


 * Rat tumor cell lines
 * GH3 (pituitary tumor)
 * 9L (glioblastoma)
 * PC12 (pheochromocytoma)


 * Mouse cell lines
 * 3T3 cells
 * MC3T3 (embryonic calvarial)
 * C3H-10T1/2 (embryonic mesenchymal)
 * NIH-3T3 (embryonic fibroblast)


 * Invertebrate cell lines
 * C6/36 Aedes albopictus (Asian tiger mosquito) larva
 * Insect cell line Sf21


 * Plant cell lines
 * Tobacco BY-2 cells (kept as cell suspension culture, they are model system of plant cell)


 * Other species cell lines
 * zebrafish ZF4 and AB9 cells.
 * Madin-Darby Canine Kidney (MDCK) epithelial cell line
 * Chinese Hamster Ovary CHO cells
 * Xenopus A6 kidney epithelial cells.

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.