Common cell lines
| | 0 commentsHuman cell lines
National Cancer Institute 60 cancer cell lines
ESTDAB database
DU145 (Prostate cancer)
Lncap (Prostate cancer)
MCF-7 (breast cancer)
MDA-MB-438 (breast cancer)
PC3 (Prostate cancer)
T47D (breast cancer)
THP-1 (acute myeloid leukemia)
U87 (glioblastoma)
SHSY5Y Human neuroblastoma cells, cloned from a myeloma
Saos-2 cells (bone cancer)
Primate cell lines
Vero (African green monkey Chlorocebus kidney epithelial cell line initiated 1962)
Rat tumor cell lines
GH3 (pituitary tumor)
PC12 (pheochromocytoma)
Mouse cell lines
MC3T3 (embryonic calvarial)
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) epithelia cell line
Xenopus A6 kidney epithelil cells
cell culture Established human cell lines
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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 Moore v. Regents of the University of California that human patients have no property rights in cell lines derived from organs removed with their consent.
Generation of hybridomas
For more details on this topic, see Hybridoma.
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 spleen (or possibly blood) of an immunised animal are combined with an immortal myeloma cell line (B cell lineage) to produce a hybridoma which has the antibody specificity of the primary lymphoctye and the immortality of the myeloma. Selective growth medium (HA or HAT) is used to select against unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.
[Applications of cell culture
Mass culture of animal cell lines is fundamental to the manufacture of viral vaccins and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic 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.
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.
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 influenza 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
For more details on this topic, see Hybridoma.
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 spleen (or possibly blood) of an immunised animal are combined with an immortal myeloma cell line (B cell lineage) to produce a hybridoma which has the antibody specificity of the primary lymphoctye and the immortality of the myeloma. Selective growth medium (HA or HAT) is used to select against unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.
[Applications of cell culture
Mass culture of animal cell lines is fundamental to the manufacture of viral vaccins and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic 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.
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.
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 influenza 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
Cell culture
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Cell culture is the process by which 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 common laboratory technique in the mid-1900s,but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.
Animal cell culture became a common laboratory technique in the mid-1900s,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 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, trypsn, 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 tumors, 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 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 for mammalian cells) 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 nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in biotechnology medical applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible, but this cannot always be accomplished.
Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture which involves growing cells in a three-dimensional environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).
Cell line cross-contamination
Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest that anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.Problems with cell line cross contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies. Major cell line repositories including the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ) have received cell line submissions from researchers that were misidentified by the researcher.Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions.ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.
To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. There are many methods for identifying cell lines including isoenzyme analysis, human lymphocyte antigen (HLA) typing and STR analysis.
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 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, trypsn, 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 tumors, 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 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 for mammalian cells) 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 nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in biotechnology medical applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible, but this cannot always be accomplished.
Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture which involves growing cells in a three-dimensional environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).
Cell line cross-contamination
Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest that anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.Problems with cell line cross contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies. Major cell line repositories including the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ) have received cell line submissions from researchers that were misidentified by the researcher.Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions.ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.
To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. There are many methods for identifying cell lines including isoenzyme analysis, human lymphocyte antigen (HLA) typing and STR analysis.
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 or senescence.
Cell-to-cell contact can stimulate cellular differentiation.
Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) and antifungals (e.g. Amphotericin B) can also be added to the growth media.
As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium in order to measure nutrient depletion.
Media changes
In the case of adherent cultures, the media can be removed directly by aspiration and replaced.
Passaging cells
Main article: Passaging
Passaging (also known as subculture 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 is commonly 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
Main article: transfection
Main article: transformation (genetics)
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 realized 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.
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 or senescence.
Cell-to-cell contact can stimulate cellular differentiation.
Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) and antifungals (e.g. Amphotericin B) can also be added to the growth media.
As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium in order to measure nutrient depletion.
Media changes
In the case of adherent cultures, the media can be removed directly by aspiration and replaced.
Passaging cells
Main article: Passaging
Passaging (also known as subculture 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 is commonly 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
Main article: transfection
Main article: transformation (genetics)
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 realized 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.
BioCyc database collection
| | 0 commentsThe BioCyc database collection is a biological database describing genome and pathway information for hundreds of organisms. It is maintained by SRI Internationl, in in Menlo Park, California.
Two databases within BioCyc are highly curated, meaning they have received extensive manual updating with information from the scientific literature. Those databases are EcoCyc and MetaCyc.
The remaining BioCyc databases were generated computationally to predict what metabolic pathways are present in these organisms. In some cases, the databases were refined manually after their generation.
BioCyc databases rely on a software system called Pathway Tools for their initial generation, subsequent updating, and for querying their content. The databases can also be installed locally.
All BioCyc databases share the same database schema, which facilitates comparisons across the databases.
Bio-fermentation technology
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- Advantages of bio-fermentation:
Standardization:
Highly controlled by scientific means during its growth and "standardized", every batch is virtually identical and, in a sense, "perfect." This is very important in pharmaceutical terms because without standardization, it is difficult or impossible to develop drug-type standards for substances like Cordyceps. Once a herb can be standardized, all kinds of studies can be conducted that will be accepted by the scientific community. However, standardization, by itself, does not make a product effective. Cost
Products can be produced more cheaply by bio-fermentation. High quality wild Cordyceps is rare and now sells for well over a thousand dollars a pound in cities like Hong Kong, Tokyo, New York or London. The culture-grown Cordyceps is available at a third of the cost, with approximately the same benefits. Fermentation technology makes this substance more readily available.
Vegetarian:
Wild Cordyceps, by weight, is mostly caterpillar. For vegetarians, and those who do not want to consume caterpillars, the new technology provides a solution. The fermentation technology does not include the caterpillar in the growing process. The fungus is grown without the use of animal nutrients and the result is a 100% pure "vegetarian" health product.
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