Cell culture

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Cell culture is the complex 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 Wikipedia:eukaryotes, especially Wikipedia:animal cells. The historical development and methods of cell culture are closely interrelated to those of Wikipedia:tissue culture and Wikipedia:organ culture.

Animal cell culture became a common Wikipedia: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[edit]

The 19th-century English physiologist Wikipedia: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 Wikipedia:Wilhelm Roux removed a portion of the Wikipedia:medullary plate of an Wikipedia:embryonic Wikipedia:chicken and maintained it in a warm Wikipedia:saline solution for several days, establishing the principle of tissue culture.[3] Wikipedia:Ross Granville Harrison, working at Wikipedia:Johns Hopkins Medical School and then at Wikipedia:Yale University, published results of his experiments from 1907–1910, establishing the methodology of Wikipedia:tissue culture.[4]

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

Concepts in mammalian cell culture[edit]

Isolation of cells[edit]

Cells can be isolated from tissues for Wikipedia: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 Wikipedia:enzymes such as collagenase, Wikipedia:trypsin, or Wikipedia:pronase, which break down the Wikipedia: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 Wikipedia: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 (called the Hayflick limit) cells undergo the process of Wikipedia: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 Wikipedia:telomerase Wikipedia:gene. There are numerous well established cell lines representative of particular cell types.

Maintaining cells in culture[edit]

Cells are grown and maintained at an appropriate Wikipedia:temperature and gas mixture (typically, 37Wikipedia:°C, 5% CO2 for mammalian cells) in a Wikipedia:cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different Wikipedia: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 Wikipedia:pH, glucose concentration, Wikipedia:growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal Wikipedia:blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with Wikipedia:viruses or Wikipedia:prions, particularly in Wikipedia: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]

Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes Wikipedia:granulosa cells exhibit estrogen production, while a higher plating density makes them appear as Wikipedia:progesterone producing Wikipedia:theca lutein cells.[6]

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 Wikipedia: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[edit]

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.[7][8][9] 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.[10][11] 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.[10][12] 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.[13] ATCC uses Wikipedia:short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.[14]

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 Wikipedia:isoenzyme analysis, Wikipedia:human lymphocyte antigen (HLA) typing and Wikipedia:STR analysis.[14]

One significant cell-line cross contaminant is the immortal Wikipedia:HeLa cell line.

Manipulation of cultured cells[edit]

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 Wikipedia:sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a Wikipedia:biosafety hood or Wikipedia:laminar flow cabinet to exclude contaminating micro-organisms. Wikipedia:Antibiotics (e.g. Wikipedia:penicillin and Wikipedia:streptomycin) and antifungals (e.g. Wikipedia:Amphotericin B) can also be added to the growth media.

As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a Wikipedia:pH indicator is added to the medium in order to measure nutrient depletion.

Media changes[edit]

In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

Passaging cells[edit]

Template:Main 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 Wikipedia:trypsin-Wikipedia: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[edit]

Template:Main Template:Main Another common method for manipulating cells involves the introduction of foreign DNA by Wikipedia:transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of Wikipedia: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 Wikipedia:viruses, in methods referred to as transduction, Wikipedia: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[edit]

One of the earliest human cell lines, descended from Wikipedia:Henrietta Lacks, who died of the cancer that those cells originated from, the cultured Wikipedia:HeLa cells shown here have been stained with Hoechst turning their nuclei blue.

Cell lines that originate with Wikipedia:humans have been somewhat controversial in Wikipedia: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 Wikipedia:Supreme Court of California held in Wikipedia: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.[15]

Generation of hybridomas[edit]

Template:Details

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 Wikipedia:spleen (or possibly blood) of an immunised animal are combined with an immortal myeloma cell line (B cell lineage) to produce a Wikipedia:hybridoma which has the antibody specificity of the primary lymphoctye and the immortality of the myeloma. Wikipedia: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[edit]

Mass culture of animal cell lines is fundamental to the manufacture of viral Wikipedia:vaccines and other products of biotechnology [16] , such as adjuvants.[17]

Culture of non-mammalian cells[edit]

Plant cell culture methods[edit]

Template:Main Template:See also Plant cell cultures are typically grown as Wikipedia: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 Wikipedia:auxin and Wikipedia:cytokinin.

Bacterial/Yeast culture methods[edit]

Template:Main

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[edit]

Template:Main The culture of Wikipedia:viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts for the growth and replication of the virus. Whole Wikipedia: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 Wikipedia:viral replication may result in host cell lysis and formation of a Wikipedia:viral plaque.

Common cell lines[edit]

Wikipedia:Human cell lines
Wikipedia:Primate cell lines
Wikipedia:Rat tumor cell lines
Wikipedia:Mouse cell lines
Plant cell lines
Other species cell lines

List of cell lines[edit]

Cell line Meaning Organism Origin tissue Morphology Link
293-T Human Kidney (embryonic) Derivative of Wikipedia:HEK 293ECACC
Wikipedia:3T3 cells "3-day transfer, inoculum 3 x 105 cells" Mouse Embryonic fibroblast Also known as NIH 3T3 ECACC
721 Human Melanoma
9L Rat Glioblastoma
A2780 Human Ovary Ovarian Cancer ECACC
A2780ADR Human Ovary Adriamycin-resistant derivative ECACC
A2780cis Human Ovary Cisplatin-resistant derivative ECACC
A172 Human glioblastoma malignant Wikipedia:glioma ECACC
A20 Murine B Wikipedia:lymphoma B Wikipedia:lymphocyte
A253 Human Head and neck Wikipedia:carcinoma Wikipedia:submandibular duct
A431 Human Skin epithelium squamous carcinoma ECACCCell Line Data Base
A-549 Human Lungcarcinoma Epithelium DSMZECACC
ALC Murine Wikipedia:bone marrow Stroma Template:PMID
B16 Murine Wikipedia:Melanoma ECCAC
B35 Rat Wikipedia:Neuroblastoma ATCC
Wikipedia:BCP-1 cells Human Wikipedia:PBMC HIV+ Lymphoma ATCC
BEAS-2B Bronchial epithelium + Adenovirus 12-SV40 virus hybrid (Ad12SV40) Human Lung Epithelial ATCC
bEnd.3 Brain endothelial Mouse Brain / Wikipedia:Cerebral cortex Endothelium ATCC
BHK-21 "Baby Wikipedia:Hamster Wikipedia:Kidney Wikipedia:Fibroblast cells" Wikipedia:Hamster Wikipedia:Kidney Wikipedia:fibroblast ECACCOlympus
BR 293 Human Breast Breast cancer
BxPC3 Biopsy xenograph of pancreatic carcinoma line 3 Human pancreatic adenocarcinoma Epithelial ATCC
C3H-10T1/2 Mouse Embryonic mesenchymal cell line ECACC
C6/36 Wikipedia:Asian tiger mosquito larval tissue ECACC
Cal-27 Human Tongue squamous cell carcinoma
CHO Chinese hamster ovary hamster Ovary Epithelium ECACCICLC
COR-L23 Human Lung ECACC
COR-L23/CPR Human Lung ECACC
COR-L23/5010 Human Lung ECACC
COR-L23/R23 Human Lung Epithelial ECACC
COS-7 Cercopithecus aethiops, origin-defective SV-40 Ape - Cercopithecus aethiops (Wikipedia:Chlorocebus) Kidney fibroblast ECACCATCC
COV-434 Human Ovary Metastatic granulosa cell carcinoma [2]ECACC
CML T1 Chronic Myelod Leukaemia T-lymphocyte 1 Human CML acute phase T cell leukaemia Blood
CMT canine mammary tumor Dog Mammary gland Epithelium
CT26 Murine Colorectal Carcinoma Colon
D17 canine Wikipedia:osteosarcoma ECACC
DH82 canine histiocytosis Wikipedia:monocyte/Wikipedia:macrophage ECACC

J Vir Meth

DU145 Human Wikipedia:Androgen insensitive Wikipedia:carcinoma Prostate
DuCaP Wikipedia:Dura mater Cancer of the Prostate Human Metastatic Prostate Cancer Epithelial Template:PMID
EL4 Mouse T cell leukaemia ECACC
EM2 Human CML blast crisis Ph+ CML line Cell Line Data Base
EM3 Human CML blast crisis Ph+ CML line Cell Line Data Base
EMT6/AR1 Mouse Breast Epithelial-like ECACC
EMT6/AR10.0 Mouse Breast Epithelial-like ECACC
FM3 Human Metastatic lymph node melanoma
Wikipedia:H1299 Human Lung Lung cancer
H69 Human Lung ECACC
HB54 Wikipedia:hybridoma Wikipedia:hybridoma secretes L243 mAb (against HLA-DR) Human Immunology
HB55 Wikipedia:hybridoma Wikipedia:hybridoma secretes MA2.1 mAb (against HLA-A2 and HLA-B17) Journal of Immunology
HCA2 Human fibroblast Journal of General Virology
HEK-293 Human embryonic kidney Human Kidney (embryonic) Epithelium ATCC
Wikipedia:HeLa Henrietta Lacks Human Wikipedia:Cervical cancer Epithelium DSMZECACC
Hepa1c1c7 clone 7 of clone 1 hepatoma line 1 Mouse Hepatoma Epithelial ECACC

ATCC

HL-60 Human leukemia Human Wikipedia:Myeloblast bloodcells ECACCDSMZ
HMEC Human mammary epithelial cell Human Epithelium ECACC
HT-29 Human Colon epithelium Adenocarcinoma ECACC

Cell Line Data Base

Jurkat Human T-Cell-Wikipedia:Leukemia white blood cells ECACC

DSMZ

Wikipedia:JY cells Human Lymphoblastoid EBV immortalised B cell
Wikipedia:K562 cells Human Lymphoblastoid CML blast crisis ECACC
Wikipedia:Ku812 Human Lymphoblastoid erythroleukemia ECACC

LGCstandards

Wikipedia:KCL22 Human Lymphoblastoid CML
Wikipedia:KG1 Human Lymphoblastoid AML
Wikipedia:KYO1 Kyoto 1 Human Lymphoblastoid CML DSMZ
LNCap Lymph node Cancer of the Prostate Human prostatic adenocarcinoma Epithelial ECACCATCC
Ma-Mel 1, 2, 3....48 Human a range of melanoma cell lines
MC-38 Mouse Adenocarcinoma
MCF-7 Michigan Cancer Foundation-7 Human Mammary gland Invasive breast ductal carcinoma ER+, PR+
MCF-10A Michigan Cancer Foundation Human mammary gland Epithelium ATCC
MDA-MB-231 M.D. Anderson - Metastatic Breast Human Breast Cancer ECACC
MDA-MB-468 M.D. Anderson - Metastatic Breast Human Breast Cancer ECACC
MDA-MB-435 M.D. Anderson - Metastatic Breast Human Breast melanoma or carcinoma (disputed) Cambridge Pathology ECACC
MDCK II Madin Darby canine kidney Dog Kidney Epithelium ECACC ATCC
MDCK II Madin Darby canine kidney Dog Kidney Epithelium [3] ATCC
MOR/0.2R Human Lung ECACC
MONO-MAC 6 Human WBC myeloid metaplasic Wikipedia:AML Cell Line Data Base
MTD-1A Mouse Epithelium
MyEnd Myocardial endothelial Mouse Endothelium
NCI-H69/CPR Human Lung ECACC
NCI-H69/LX10 Human Lung ECACC
NCI-H69/LX20 Human Lung ECACC
NCI-H69/LX4 Human Lung ECACC
Wikipedia:NIH-3T3 NIH, 3-day transfer, inoculum 3 x 105 cells Mouse embryo fibroblast ECACCATCC
NALM-1 peripheral blood blast-crisis CML Cancer Genetics and Cytogenetics
NW-145 Melanoma ESTDAB
OPCN / OPCT cell lines Onyvax [4] Prostate Cancer.... Range of prostate tumour lines Asterand
Peer Human T cell leukemia DSMZ
PNT-1A / PNT 2 Prostate tumour lines ECACC
RenCa Renal Carcinoma Mouse renal carcinoma
RIN-5F Mouse Pancreas
RMA/RMAS Mouse T cell tumour
Wikipedia:Saos-2 cells Human Osteosarcoma ECACC
Sf-9 Spodoptera frugiperda insect - Spodoptera frugiperda (moth) Ovary DSMZECACC
SkBr3 Human Breast carcinoma
T2 Human T cell leukemia/B cell line hybridoma DSMZ
T-47D Human Mammary gland ductal carcinoma
T84 Human colorectal Carcinoma / Lungmetastasis Epithelium ECACCATCC
Wikipedia:THP1 cell line Human Monocyte AML ECACC
U373 Human Glioblastoma-astrocytoma Epithelium
U87 Human glioblastoma-astrocytoma Epithelial-like Abcam
U937 Human Leukaemic monocytic lymphoma ECACC
VCaP Vertebra Prostate Cancer Human Metastatic prostate cancer Epithelial ECACC ATCC
Wikipedia:Vero cells 'Vera Reno' ('Green kidney') / 'Vero' ('truth') African Green Monkey Kidney epithelium ECACC
WM39 Human skin Primary melanoma
WT-49 Human Lymphoblastoid
X63 Mouse Melanoma
YAC-1 Mouse Lymphoma Cell Line Data Base ECACC
YAR Human B-cell EBV transofrmed [5] Human Immunology

Note: this list is a sample of available cell lines, and is not comprehensive

See also[edit]

References and notes[edit]

  1. ""Cell Culture"". http://www.bioteach.ubc.ca/Bioengineering/CellCulture/index.htm. Retrieved on 2006-04-19. 
  2. ""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 on 2006-04-19. 
  3. ""Animals and alternatives in testing."". http://caat.jhsph.edu/pubs/animal_alts/appendix_c.htm. Retrieved on 2006-04-19. 
  4. Schiff, Judith Ann. ""An unsung hero of medical research."". http://www.yalealumnimagazine.com/issues/02_02/old_yale.html. Retrieved on 2006-04-19.  Wikipedia:Yale Alumni Magazine, February 2002.
  5. "LipiMAX purified lipoprotein solution from bovine serum". Selborne Biological Services. 2006. http://www.selbornebiological.com/products/lipimax.htm. Retrieved on 2010-02-02. 
  6. Portela VM, Zamberlam G, Price CA (April 2010). "Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells". Fertil. Steril. 93 (6): 2050–5. doi:10.1016/j.fertnstert.2009.01.151. PMID 19324349. 
  7. Drexler, HG; Dirks, WG; Macleod, RA (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. 
  8. Drexler, HG; Macleod, RA; Dirks, WG (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. 
  9. 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. 
  10. 10.0 10.1 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. 
  11. Liscovitch, M; Ravid, D (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. 
  12. Macleod, RA; Dirks, WG; Matsuo, Y; Kaufmann, M; Milch, H; Drexler, HG (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. doi:10.1002/(SICI)1097-0215(19991112)83:4<555::AID-IJC19>3.0.CO;2-2. ISSN 0020-7136. PMID 10508494. 
  13. 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. 
  14. 14.0 14.1 Dunham, J.H. and Guthmiller, P. (2008) Doing good science: Authenticating cell line identity. Cell Notes 22, 15–17.
  15. Ceb.com
  16. | author = 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. | year = 2006 | month = February | title = Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization | journal = Journal of Virology | volume = 80 | issue = 4 | pages = 1959–1964 | publisher = American Society for Microbiology | location = United States | issn = 0022-538X | doi = 10.1128/JVI.80.4.1959-1964.2006 | url = http://jvi.asm.org/cgi/content/abstract/80/4/1959 | accessdate = 2010-01-31 }}
  17. "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 on 2010-01-31. 

External links[edit]

Endotoxin free water for cell cultures

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