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Cell culture

From Molecular Biology Wiki

Cell culture is the technique of growing and maintaining the cells of multicellular organisms outside of the organism in specially designed containers, which are located in conditions which attempt to mimic the precise environmental conditions such as temperature, humidity, nutrition, and contamination-free conditions that were present in that organism.


Cells, tissues, and organs that are isolated and maintained in a laboratory setting are the living objects that are "cultured". These cells can prokaryotic or eukaryotic cells in origin although in research, the term "cell culture" has come to refer mainly to the culturing of cells from animals and humans, namely eukaryotic cells.

The culture of bacteria is often referred to as bacterial culture and not cell culture.

The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.


Contents

Purpose of Cell Culture

Cell culture allows the study of cells under controlled conditions. This allows researchers to examine the effects of specific conditions and mutations on cell physiology.

  • Cell culture allows the investigatation of normal cell physiology and biochemistry of cells. Studies of cell metabolism and the cell cycle.
  • It allows researchers to examine the effect of various chemical compounds or drugs on a given cell types. For example researchers can examine the toxicity of a certain chemical in normal cells. Also, the ability of a drug to kill cancer cells can be also tested in cell culture.

Limitations of Cell Culture

Cell culture is referred to as an ex vivo study of the cellular milleu. This is a problem because the cell is not in its normal physiological and original environment. Cell culture is simply an attempt to provide a simulated environment. "The study of cells in cell culture is akin to studying polar bear habits at the local zoo." (quote from Moleculardude)

Also a problem in cell culture is the usage of cells which are transformed. HepG2 cells for example are derived from a liver cancer cell line from humans, and thus are a hepatocarcinoma cell line. The disadvantage of this is these cells often differ markedly from normal cells in that they have altered or a loss of their specific cell function due to mutations.

Alternatives to using transformed cells is Primary cell culture, which is the culture of primary cells, which are untransformed, normal cells isolated recently from an animal by tissue culture.


Missing Features in Cell Culture

  • Cell culture is missing the original blood circulation.
  • Cell culture often is missing the original tissue organisation and structure
  • Factors in the blood such as hormones are often missing.
  • Cells are often not always and entirely in contact with other cells, as cultures are never left to 100% confluency. This is a problem as normal cells (primary cells) need cell to cell contacts. Transformed cells usually can grow and divide quite well in the absence of cell-cell contacts.


Also of note is hormones are usually added at very high concentrations to treat cells and are not usually physiological.

Problems with Cell Culture

Cell culture has many problems including Cell culture contamination by bacteria, mycoplasma, and fungus.

Also, cells must be sub-cultured frequently to prevent over-crowding of cells.

Furthermore, media must often be change in order to prevent the build up of contaminants, toxns and provide fresh nutrients as nutrients are used up quickly by quickly growing cells (and in culture these are often transformed, quickly growing cells).

History of Cell culture

Animal cell culture only became a routine laboratory technique in the 1950s,[1] but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.[2]


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.[1] 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.[3] 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.[4]

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 in mammalian cell culture

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 cells are 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 an animal or person 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.


Sub-culture of cells

see Cell subculture.


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
  • Cell-to-cell contact can stimulate promicuous 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 biproducts 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

Main article: transfection

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.

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. [5]

It is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be.[6] 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. [7]

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. 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), 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.

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,[8] [9] or the use of adjuvants. [10]

Culture of non-mammalian cells

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

Main article: microbiological culture

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 as hosts for the growth and replication of the virus. Viruses infect cells, causing them to lyse and form a viral plaque.

Common cell lines

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

See also

References and notes

  1. Schiff, Judith Ann. Yale Alumni Magazine, February 2002.
  2. ceb
  3. (MacLeoud et al. 1999)
  4. (Masters 2002). See List of contaminated cell lines.
  5. cold as vector news
  6. cold as vector research abstract
  7. adjuvant

fr:culture cellulaire it:Coltura cellulare pl:Kultura komórkowa simple:Cell culture sv:Cellodling

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