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,
[1] but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.
[2]
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.
[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.
[edit] Concepts in mammalian cell culture
[edit] 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 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
expression of the
telomerase gene. There are numerous well established cell lines representative of particular cell types.
[edit] 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. Alternative strategies involve sourcing the animal blood from countries with minimum
BSE/
TSE risk such as Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.
[5]
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).
[edit] 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.
[6][7][8] 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.
[9][10] 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.
[9][11] 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.
[12] ATCC uses
short tandem repeat (STR)
DNA fingerprinting to authenticate its cell lines.
[13]
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.
[13]
One significant cell-line cross contaminant is the immortal
HeLa cell line.
[edit] 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:
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.
[edit] Media changes
In the case of adherent cultures, the media can be removed directly by aspiration and replaced.
[edit] Passaging cells
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.
[edit] Transfection and transduction
Main article:
transfectionAnother 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.
[edit] Established human cell lines
One of the earliest human cell lines, descended from
Henrietta Lacks, who died of the cancer that those cells originated from, the cultured
HeLa cells shown here have been stained with
Hoechst turning their
nuclei blue.
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.
[14]
[edit] 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.
[edit] 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, 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, use of single embryonic cell and
somatic embryos as a source for direct gene transfer via practicle bombardment, transit
gene expression and
confocal microscopy observation is one of its applications. It also offers to confirm single cell origin of somatic embryos and the asymmetry of the first cell division, which starts the process.
[edit] 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. The major application of human cell culture is in stem cell industry where
mesenchymal stem cells can be cultured and cryopreserved for future use.
[edit] 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,
[15][16] or the use of
adjuvants.
[17]
[edit] Culture of non-mammalian cells
[edit] 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.
[edit] 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.
[edit] Viral culture methods
Main article:
Viral cultureThe 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. Whole
wild 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.
[edit] Common cell lines
- Human cell lines
- Primate cell lines
- Rat tumor cell lines
- Mouse cell lines
- Plant cell lines
- Other species cell lines
[edit] List of cell lines
Note: this list is a sample of available cell lines, and is not comprehensive
[edit] See also
[edit] References and notes
- ^ ""Cell Culture"". http://www.bioteach.ubc.ca/Bioengineering/CellCulture/index.htm. Retrieved 2006-04-19.
- ^ ""Some landmarks in the development of tissue and cell culture."". http://www.ncbi.nlm.nih.gov/books/bv.fcgi?db=Books&rid=mboc4.table.1516. Retrieved 2006-04-19.
- ^ ""Animals and alternatives in testing."". http://caat.jhsph.edu/pubs/animal_alts/appendix_c.htm. Retrieved 2006-04-19.
- ^ Schiff, Judith Ann. ""An unsung hero of medical research."". http://www.yalealumnimagazine.com/issues/02_02/old_yale.html. Retrieved 2006-04-19. Yale Alumni Magazine, February 2002.
- ^ "LipiMAX purified lipoprotein solution from bovine serum". Selborne Biological Services. 2006. http://www.selbornebiological.com/products/lipimax.htm. Retrieved 2010-02-02.
- ^ Drexler, HG; Dirks; Macleod (Oct 1999). "False human hematopoietic cell lines: cross-contaminations and misinterpretations". Leukemia 13 (10): 1601–7. doi:10.1038/sj/leu/2401510. ISSN 0887-6924. PMID 10516762.
- ^ Drexler, HG; Macleod; Dirks (Dec 2001). "Cross-contamination: HS-Sultan is not a myeloma but a Burkitt lymphoma cell line" (Free full text). Blood 98 (12): 3495–6. doi:10.1182/blood.V98.12.3495. ISSN 0006-4971. PMID 11732505. http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=11732505.
- ^ Cabrera, CM; Cobo, F; Nieto, A; Cortés, JL; Montes, RM; Catalina, P; Concha, A (Jun 2006). "Identity tests: determination of cell line cross-contamination". Cytotechnology 51 (2): 45–50. doi:10.1007/s10616-006-9013-8. ISSN 0920-9069. PMID 19002894.
- ^ a b Chatterjee, R (Feb 2007). "Cell biology. Cases of mistaken identity.". Science (New York, N.Y.) 315 (5814): 928–31. doi:10.1126/science.315.5814.928. ISSN 0036-8075. PMID 17303729.
- ^ Liscovitch, M; Ravid (Jan 2007). "A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells.". Cancer letters 245 (1-2): 350–2. doi:10.1016/j.canlet.2006.01.013. ISSN 0304-3835. PMID 16504380.
- ^ Macleod, RA; Dirks; Matsuo; Kaufmann; Milch; Drexler (Nov 1999). "Widespread intraspecies cross-contamination of human tumor cell lines arising at source.". International journal of cancer. Journal international du cancer 83 (4): 555–63. ISSN 0020-7136. PMID 10508494.
- ^ Masters, JR (Apr 2002). "HeLa cells 50 years on: the good, the bad and the ugly.". Nature reviews. Cancer 2 (4): 315–9. doi:10.1038/nrc775. ISSN 1474-175X. PMID 12001993.
- ^ a b Dunham, J.H. and Guthmiller, P. (2008) Doing good science: Authenticating cell line identity. Cell Notes 22, 15–17.
- ^ Ceb.com
- ^ Reuters (2006-01-26). Wired.com "Quickie Bird Flu Vaccine Created". Wired. http://wired.com/news/wireservice/0,70102-0.html?tw=wn_index_7 Wired.com. Retrieved 2010-01-31.
- ^ Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, Robbins PD, Swayne DE, Donis RO, Katz JM, Barratt-Boyes SM, Gambotto A. (February 2006). "Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization". Journal of Virology (United States: American Society for Microbiology) 80 (4): 1959–1964. doi:10.1128/JVI.80.4.1959-1964.2006. ISSN 0022-538X. http://jvi.asm.org/cgi/content/abstract/80/4/1959. Retrieved 2010-01-31.
- ^ "NIAID Taps Chiron to Develop Vaccine Against H9N2 Avian Influenza". National Institute of Allergy and Infectious Diseases (NIAID). 2004-08-17. http://www3.niaid.nih.gov/news/newsreleases/2004/h9n2.htm. Retrieved 2010-01-31.
[edit] External links
