cell culture

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 the body.{{cite web|url=http://www.whonamedit.com/synd.cfm/2119.html|title=Whonamedit - Ringer's solution|publisher=whonamedit.com|access-date=2014-06-09}} In 1885 Wilhelm Roux removed a section of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the basic principle of tissue culture. In 1907 the zoologist Ross Granville Harrison demonstrated the growth of frog embryonic cells that would give rise to nerve cells in a medium of clotted lymph. In 1913, E. Steinhardt, C. Israeli, and R. A. Lambert grew vaccinia virus in fragments of guinea pig corneal tissue.{{Cite journal | vauthors = Steinhardt E, Israeli C, Lambert RA |date=1913 |title=Studies on the Cultivation of the Virus of Vaccinia |journal=The Journal of Infectious Diseases |volume=13 |issue=2 |pages=294–300 |doi=10.1093/infdis/13.2.294 |issn=0022-1899 |jstor=30073371}} In 1996, the first use of regenerative tissue was used to replace a small length of urethra, which led to the understanding that the technique of obtaining samples of tissue, growing it outside the body without a scaffold, and reapplying it, can be used for only small distances of less than 1 cm.{{cite web | vauthors = Atala A |title=Growing new organs |url=https://www.ted.com/talks/anthony_atala_growing_new_organs |work=TEDMED |year=2009 |language=en |access-date=2021-08-23}}{{cite web|url=http://caat.jhsph.edu/pubs/animal_alts/appendix_c.htm|title=Animals and alternatives in testing|access-date=2006-04-19|archive-url = https://web.archive.org/web/20060225204205/http://caat.jhsph.edu/pubs/animal_alts/appendix_c.htm |archive-date = 2006-02-25}}{{cite journal | vauthors = Fentem JH | title = Working together to respond to the challenges of EU policy to replace animal testing | journal = Alternatives to Laboratory Animals | volume = 34 | issue = 1 | pages = 11–18 | date = February 2006 | pmid = 16522146 | doi = 10.1177/026119290603400116 | s2cid = 10339716 | doi-access = free }} Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907 to 1910, establishing the methodology of tissue culture.{{cite web | vauthors = Schiff JA | title = An unsung hero of medical research | url = http://www.yalealumnimagazine.com/issues/02_02/old_yale.html | access-date = 2006-04-19 | work = Yale Alumni Magazine | date = February 2002 | archive-date = 2012-11-14 | archive-url = https://web.archive.org/web/20121114035855/http://yalealumnimagazine.com/issues/02_02/old_yale.html | url-status = dead }}

Gottlieb Haberlandt first pointed out the possibilities of the culture of isolated tissues, plant tissue culture.{{cite journal | vauthors = Bonner J | title = Plant Tissue Cultures from a Hormone Point of View | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 22 | issue = 6 | pages = 426–430 | date = June 1936 | pmid = 16588100 | pmc = 1076796 | doi = 10.1073/pnas.22.6.426 | doi-access = free | jstor = 86579 | bibcode = 1936PNAS...22..426B }} He suggested that the potentialities of individual cells via tissue culture as well as that the reciprocal influences of tissues on one another could be determined by this method. Since Haberlandt's original assertions, methods for tissue and cell culture have been realized, leading to significant discoveries in biology and medicine. He presented his original idea of totipotentiality in 1902, stating that "Theoretically all plant cells are able to give rise to a complete plant."Haberlandt, G. (1902) Kulturversuche mit isolierten Pflanzenzellen. Sitzungsber. Akad. Wiss. Wien. Math.-Naturwiss. Kl., Abt. J. 111, 69–92.{{cite journal | vauthors = Noé AC | title = Gottlieb Haberlandt | journal = Plant Physiology | volume = 9 | issue = 4 | pages = 850–855 | date = October 1934 | pmid = 16652925 | pmc = 439112 | doi = 10.1104/pp.9.4.850 }}[https://www.springer.com/life+sciences/plant+sciences/book/978-3-211-83839-6 Plant Tissue Culture]. 100 years since Gottlieb Haberlandt. Laimer, Margit; Rücker, Waltraud (Eds.) 2003. Springer {{ISBN|978-3-211-83839-6}} The term tissue culture was coined by American pathologist Montrose Thomas Burrows.{{cite journal | vauthors = Carrel A, Burrows MT | title = Cultivation of Tissues in Vitro and ITS Technique | journal = The Journal of Experimental Medicine | volume = 13 | issue = 3 | pages = 387–396 | date = March 1911 | pmid = 19867420 | pmc = 2125263 | doi = 10.1084/jem.13.3.387 }}

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 injectable polio vaccine developed by 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 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. Cell culture has contributed to the development of vaccines for many diseases.

File:Cho_cells_adherend2.jpg]]

In modern usage, "tissue culture" generally refers to the growth of cells from a tissue from a multicellular organism in vitro. These cells may be cells isolated from a donor organism (primary cells) or an immortalised cell line. The cells are bathed in a culture medium, which contains essential nutrients and energy sources necessary for the cells' survival.{{Cite book | vauthors = Martin BM |url=https://books.google.com/books?id=tl3dBwAAQBAJ |title=Tissue Culture Techniques: An Introduction |date=2013-12-01 |publisher=Springer Science & Business Media |isbn=978-1-4612-0247-9 |pages=29–30 |language=en}} Thus, in its broader sense, "tissue culture" is often used interchangeably with "cell culture". On the other hand, the strict meaning of "tissue culture" refers to the culturing of tissue pieces, i.e. explant culture.

Tissue culture is an important tool for the study of the biology of cells from multicellular organisms. It provides an in vitro model of the tissue in a well defined environment which can be easily manipulated and analysed. In animal tissue culture, cells may be grown as two-dimensional monolayers (conventional culture) or within fibrous scaffolds or gels to attain more naturalistic three-dimensional tissue-like structures (3D culture). A 1988 NIH SBIR grant report showed that electrospinning could be used to produce nano- and submicron-scale polymeric fibrous scaffolds specifically intended for use as in vitro cell and tissue substrates. This early use of electrospun fibrous lattices for cell culture and tissue engineering showed that various cell types would adhere to and proliferate upon polycarbonate fibers. It was noted that as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more rounded 3-dimensional morphology generally observed of tissues in vivo.{{Cite web | vauthors = Simon EM |date=1988 |title=Phase I Final Report: Fibrous Substrates for Cell Culture (R3RR03544A) |url= https://www.researchgate.net/publication/317053872 |access-date=2017-05-22 |website=ResearchGate |language=en}}

Plant tissue culture in particular is concerned with the growing of entire plants from small pieces of plant tissue, cultured in medium.Urry, L. A., Campbell, N. A., Cain, M. L., Reece, J. B., Wasserman, S. (2007). Biology. United Kingdom: Benjamin-Cummings Publishing Company. p. 860

{{Main|Cell isolation}}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. Cells can be isolated from solid tissues by digesting the extracellular matrix using enzymes such as collagenase, trypsin, or pronase, before agitating the tissue to release the cells into suspension.{{cite journal | vauthors = Voigt N, Pearman CM, Dobrev D, Dibb KM | title = Methods for isolating atrial cells from large mammals and humans | journal = Journal of Molecular and Cellular Cardiology | volume = 86 | pages = 187–198 | date = September 2015 | pmid = 26186893 | doi = 10.1016/j.yjmcc.2015.07.006 | doi-access = free }}{{cite journal | vauthors = Louch WE, Sheehan KA, Wolska BM | title = Methods in cardiomyocyte isolation, culture, and gene transfer | journal = Journal of Molecular and Cellular Cardiology | volume = 51 | issue = 3 | pages = 288–298 | date = September 2011 | pmid = 21723873 | pmc = 3164875 | doi = 10.1016/j.yjmcc.2011.06.012 }} 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.

An established or immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene.

Numerous cell lines are well established as representative of particular cell types.

For the majority of isolated primary cells, they undergo the process of senescence and stop dividing after a certain number of population doublings while generally retaining their viability (described as the Hayflick limit).

File:DMEM cell culture medium.jpg

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the cell 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 the serum of animal blood, such as fetal bovine serum (FBS), bovine calf serum, equine serum, and porcine serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in medical biotechnology applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible and use human platelet lysate (hPL).Hemeda, H., Giebel, B., Wagner, W. (16Feb2014) Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells Cytotherapy p170-180 issue 2 doi.10.1016 This eliminates the worry of cross-species contamination when using FBS with human cells. hPL has emerged as a safe and reliable alternative as a direct replacement for FBS or other animal serum. In addition, chemically defined media can be used to eliminate any serum trace (human or animal), but this cannot always be accomplished with different cell types. Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk, such as The United States, Australia and New Zealand,{{cite web|url=http://www.bovalco.com/blog/post/view/9|title=Post - Blog | Boval BioSolutions, LLC|publisher=bovalco.com|access-date=2014-12-02|archive-date=10 September 2014|archive-url=https://web.archive.org/web/20140910200039/http://www.bovalco.com/blog/post/view/9|url-status=dead}} and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.{{cite web|url=http://www.selbornebiological.com/products/lipimax.htm|title=LipiMAX purified lipoprotein solution from bovine serum|year=2006|work=Selborne Biological Services|access-date=2010-02-02|archive-date=2012-07-19|archive-url=https://web.archive.org/web/20120719014420/http://www.selbornebiological.com/products/lipimax.htm|url-status=dead}}

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 granulosa cells exhibit estrogen production, while a higher plating density makes them appear as progesterone-producing theca lutein cells.{{cite journal | vauthors = Portela VM, Zamberlam G, Price CA | title = Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells | journal = Fertility and Sterility | volume = 93 | issue = 6 | pages = 2050–2055 | date = April 2010 | pmid = 19324349 | doi = 10.1016/j.fertnstert.2009.01.151 | doi-access = free }}

Cells can be grown either in suspension or adherent cultures.{{cite journal | vauthors = Jaccard N, Macown RJ, Super A, Griffin LD, Veraitch FS, Szita N | title = Automated and online characterization of adherent cell culture growth in a microfabricated bioreactor | journal = Journal of Laboratory Automation | volume = 19 | issue = 5 | pages = 437–443 | date = October 2014 | pmid = 24692228 | pmc = 4230958 | doi = 10.1177/2211068214529288 }} 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 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 (such as collagen and laminin) 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 (3-D) 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).{{cite journal | vauthors = Humpel C | title = Organotypic brain slice cultures: A review | journal = Neuroscience | volume = 305 | pages = 86–98 | date = October 2015 | pmid = 26254240 | pmc = 4699268 | doi = 10.1016/j.neuroscience.2015.07.086 }}

There are different kinds of cell culture media which being used routinely in life science including the following:

• MEM
• DMEM
• RPMI 1640
• Ham's f-12
• IMDM
• Leibovitz L-15
• DMEM/F-12
• GMEM

{{Main|List of contaminated cell lines}}

Cell line cross-contamination can be a problem for scientists working with cultured cells.{{cite journal | vauthors = Neimark J | title = Line of attack | journal = Science | volume = 347 | issue = 6225 | pages = 938–940 | date = February 2015 | pmid = 25722392 | doi = 10.1126/science.347.6225.938 | bibcode = 2015Sci...347..938N | doi-access = free }} Studies suggest anywhere from 15 to 20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.{{cite journal | vauthors = Drexler HG, Dirks WG, MacLeod RA | title = False human hematopoietic cell lines: cross-contaminations and misinterpretations | journal = Leukemia | volume = 13 | issue = 10 | pages = 1601–1607 | date = October 1999 | pmid = 10516762 | doi = 10.1038/sj.leu.2401510 | doi-access = free }}{{cite journal | vauthors = Drexler HG, MacLeod RA, Dirks WG | title = Cross-contamination: HS-Sultan is not a myeloma but a Burkitt lymphoma cell line | journal = Blood | volume = 98 | issue = 12 | pages = 3495–3496 | date = December 2001 | pmid = 11732505 | doi = 10.1182/blood.V98.12.3495 | doi-access = free }}{{cite journal | vauthors = Cabrera CM, Cobo F, Nieto A, Cortés JL, Montes RM, Catalina P, Concha A | title = Identity tests: determination of cell line cross-contamination | journal = Cytotechnology | volume = 51 | issue = 2 | pages = 45–50 | date = June 2006 | pmid = 19002894 | pmc = 3449683 | doi = 10.1007/s10616-006-9013-8 }} 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.{{cite journal | vauthors = Chatterjee R | title = Cell biology. Cases of mistaken identity | journal = Science | volume = 315 | issue = 5814 | pages = 928–931 | date = February 2007 | pmid = 17303729 | doi = 10.1126/science.315.5814.928 | s2cid = 13255156 }}{{cite journal | vauthors = Liscovitch M, Ravid D | title = 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 | journal = Cancer Letters | volume = 245 | issue = 1–2 | pages = 350–352 | date = January 2007 | pmid = 16504380 | doi = 10.1016/j.canlet.2006.01.013 }} Major cell line repositories, including the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that were misidentified by them.{{cite journal | vauthors = MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG | title = Widespread intraspecies cross-contamination of human tumor cell lines arising at source | journal = International Journal of Cancer | volume = 83 | issue = 4 | pages = 555–563 | date = November 1999 | pmid = 10508494 | doi = 10.1002/(SICI)1097-0215(19991112)83:4<555::AID-IJC19>3.0.CO;2-2 | doi-access = free }} 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.{{cite journal | vauthors = Masters JR | title = HeLa cells 50 years on: the good, the bad and the ugly | journal = Nature Reviews. Cancer | volume = 2 | issue = 4 | pages = 315–319 | date = April 2002 | pmid = 12001993 | doi = 10.1038/nrc775 | s2cid = 991019 }} ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.{{cite journal | vauthors = Dunham JH, Guthmiller P | year = 2008 | title = Doing good science: Authenticating cell line identity | url = http://www.promega.com/cnotes/cn022/cn022_15.pdf | journal = Cell Notes | volume = 22 | pages = 15–17 | access-date = 2008-10-28 | archive-url = https://web.archive.org/web/20081028200822/http://www.promega.com/cnotes/cn022/cn022_15.pdf | archive-date = 2008-10-28 | url-status = dead }}

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. Many methods are used to identify cell lines, including isoenzyme analysis, human lymphocyte antigen (HLA) typing, chromosomal analysis, karyotyping, morphology and STR analysis.

One significant cell-line cross contaminant is the immortal HeLa cell line. HeLa contamination was first noted in the early 1960s in non-human culture in the USA. Intraspecies contamination was discovered in nineteen cell lines in the seventies. In 1974, five human cell lines from the Soviet Union were found to be HeLa. A follow-up study analysing 50-odd cell lines indicated that half had HeLa markers, but contaminant HeLa had hybridised with the original cell lines. HeLa cell contamination from air droplets has been reported. HeLa was even unknowingly injected into human subjects by Jonas Salk in a 1978 vaccine trial.Brendan P. Lucey, Walter A. Nelson-Rees, Grover M. Hutchins; Henrietta Lacks, HeLa Cells, and Cell Culture Contamination. Arch Pathol Lab Med 1 September 2009; 133 (9): 1463–1467. doi: https://doi.org/10.5858/133.9.1463

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
• Changes in pH of 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 cellular differentiation.
• Genetic and epigenetic alterations, with a natural selection of the altered cells potentially leading to overgrowth of abnormal, culture-adapted cells with decreased differentiation and increased proliferative capacity.{{cite journal | vauthors = Nguyen HT, Geens M, Spits C | title = Genetic and epigenetic instability in human pluripotent stem cells | journal = Human Reproduction Update | volume = 19 | issue = 2 | pages = 187–205 | year = 2012 | pmid = 23223511 | doi = 10.1093/humupd/dms048 | doi-access = free }}

The choice of culture medium might affect the physiological relevance of findings from cell culture experiments due to the differences in the nutrient composition and concentrations.{{cite journal | vauthors = Lagziel S, Gottlieb E, Shlomi T | title = Mind your media | journal = Nature Metabolism | volume = 2 | issue = 12 | pages = 1369–1372 | date = December 2020 | pmid = 33046912 | doi = 10.1038/s42255-020-00299-y | s2cid = 222319735 }} A systematic bias in generated datasets was recently shown for CRISPR and RNAi gene silencing screens,{{cite journal | vauthors = Lagziel S, Lee WD, Shlomi T | title = Inferring cancer dependencies on metabolic genes from large-scale genetic screens | journal = BMC Biology | volume = 17 | issue = 1 | pages = 37 | date = April 2019 | pmid = 31039782 | pmc = 6489231 | doi = 10.1186/s12915-019-0654-4 | doi-access = free }} and for metabolic profiling of cancer cell lines. Using a growth medium that better represents the physiological levels of nutrients can improve the physiological relevance of in vitro studies and recently such media types, as Plasmax{{cite journal | vauthors = Vande Voorde J, Ackermann T, Pfetzer N, Sumpton D, Mackay G, Kalna G, Nixon C, Blyth K, Gottlieb E, Tardito S | display-authors = 6 | title = Improving the metabolic fidelity of cancer models with a physiological cell culture medium | journal = Science Advances | volume = 5 | issue = 1 | pages = eaau7314 | date = January 2019 | pmid = 30613774 | pmc = 6314821 | doi = 10.1126/sciadv.aau7314 | bibcode = 2019SciA....5.7314V }} and Human Plasma Like Medium (HPLM),{{cite journal | vauthors = Cantor JR, Abu-Remaileh M, Kanarek N, Freinkman E, Gao X, Louissaint A, Lewis CA, Sabatini DM | display-authors = 6 | title = Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase | journal = Cell | volume = 169 | issue = 2 | pages = 258–272.e17 | date = April 2017 | pmid = 28388410 | pmc = 5421364 | doi = 10.1016/j.cell.2017.03.023 }} were developed.

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 aseptic technique. Aseptic technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety cabinet or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) and antifungals (e.g.amphotericin B and Antibiotic-Antimycotic solution) 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 to measure nutrient depletion.

In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced. Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media.

{{main|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. Some cell cultures, such as RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers.

{{main|Transfection|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 gene 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.

File:HeLa cells stained with Hoechst 33258.jpg cells have been stained with Hoechst turning their nuclei blue, and are one of the earliest human cell lines descended from Henrietta Lacks, who died of cervical cancer from which these cells originated.]]

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.{{cite web|url=http://online.ceb.com/calcases/C3/51C3d120.htm |title=Moore v. Regents of University of California (1990) 51 C3d 120 |publisher=Online.ceb.com |access-date=2012-01-27}}

{{further|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 lymphocyte 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.

A cell strain is derived either from a primary culture or a cell line by the selection or cloning of cells having specific properties or characteristics which must be defined. Cell strains are cells that have been adapted to culture but, unlike cell lines, have a finite division potential. Non-immortalized cells stop dividing after 40 to 60 population doublings{{cite journal | vauthors = Hayflick L | title = A brief history of the mortality and immortality of cultured cells | journal = The Keio Journal of Medicine | volume = 47 | issue = 3 | pages = 174–182 | date = September 1998 | pmid = 9785764 | doi = 10.2302/kjm.47.174 | series = 3 | doi-access = free }} and, after this, they lose their ability to proliferate (a genetically determined event known as senescence).{{cite web|title=Worthington tissue guide|url=http://www.worthington-biochem.com/tissuedissociation/glossary.html|access-date=2013-04-30}}

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology. Culture of human stem cells is used to expand the number of cells and differentiate the cells into various somatic cell types for transplantation.{{cite journal | vauthors = Qian L, Saltzman WM | title = Improving the expansion and neuronal differentiation of mesenchymal stem cells through culture surface modification | journal = Biomaterials | volume = 25 | issue = 7–8 | pages = 1331–1337 | year = 2004 | pmid = 14643607 | doi = 10.1016/j.biomaterials.2003.08.013 }} Stem cell culture is also used to harvest the molecules and exosomes that the stem cells release for the purposes of therapeutic development.{{cite journal | vauthors = Maguire G | title = Therapeutics from Adult Stem Cells and the Hype Curve | journal = ACS Medicinal Chemistry Letters | volume = 7 | issue = 5 | pages = 441–443 | date = May 2016 | pmid = 27190588 | pmc = 4867479 | doi = 10.1021/acsmedchemlett.6b00125 }}

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. Mammalian cells ensure expressed proteins are folded correctly and possess human-like glycosylation and post-translational modifications.{{Cite journal |last1=Mark |first1=Jacqueline Kar Kei |last2=Lim |first2=Crystale Siew Ying |last3=Nordin |first3=Fazlina |last4=Tye |first4=Gee Jun |date=2022-11-01 |title=Expression of mammalian proteins for diagnostics and therapeutics: a review |journal=Molecular Biology Reports |language=en |volume=49 |issue=11 |pages=10593–10608 |doi=10.1007/s11033-022-07651-3 |issn=1573-4978 |pmc=9175168 |pmid=35674877}} 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 particle 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.

Cell culture is also a key technique for cellular agriculture, which aims to provide both new products and new ways of producing existing agricultural products like milk, (cultured) meat, fragrances, and rhino horn from cells and microorganisms. It is therefore considered one means of achieving animal-free agriculture. It is also a central tool for teaching cell biology.{{cite journal | vauthors = Prieto D, Aparicio G, Sotelo-Silveira JR | title = Cell migration analysis: A low-cost laboratory experiment for cell and developmental biology courses using keratocytes from fish scales | journal = Biochemistry and Molecular Biology Education | volume = 45 | issue = 6 | pages = 475–482 | date = November 2017 | pmid = 28627731 | doi = 10.1002/bmb.21071 | doi-access = free }}

Research in tissue engineering, stem cells and molecular biology primarily involves cultures of cells on flat plastic dishes. This technique is known as two-dimensional (2D) cell culture, and was first developed by Wilhelm Roux who, in 1885, removed a portion of the medullary plate of an embryonic chicken and maintained it in warm saline for several days on a flat glass plate. From the advance of polymer technology arose today's standard plastic dish for 2D cell culture, commonly known as the Petri dish. Julius Richard Petri, a German bacteriologist, is generally credited with this invention while working as an assistant to Robert Koch. Various researchers today also utilize culturing laboratory flasks, conicals, and even disposable bags like those used in single-use bioreactors.

Aside from Petri dishes, scientists have long been growing cells within biologically derived matrices such as collagen or fibrin, and more recently, on synthetic hydrogels such as polyacrylamide or PEG. They do this in order to elicit phenotypes that are not expressed on conventionally rigid substrates. There is growing interest in controlling matrix stiffness,{{cite journal | vauthors = Discher DE, Janmey P, Wang YL | title = Tissue cells feel and respond to the stiffness of their substrate | journal = Science | volume = 310 | issue = 5751 | pages = 1139–1143 | date = November 2005 | pmid = 16293750 | doi = 10.1126/science.1116995 | s2cid = 9036803 | citeseerx = 10.1.1.318.690 | bibcode = 2005Sci...310.1139D }} a concept that has led to discoveries in fields such as:

• Stem cell self-renewal{{cite journal | vauthors = Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, Nguyen NK, Thrun S, Lutolf MP, Blau HM | display-authors = 6 | title = Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture | journal = Science | volume = 329 | issue = 5995 | pages = 1078–1081 | date = August 2010 | pmid = 20647425 | pmc = 2929271 | doi = 10.1126/science.1191035 | bibcode = 2010Sci...329.1078G }}{{cite journal | vauthors = Chowdhury F, Li Y, Poh YC, Yokohama-Tamaki T, Wang N, Tanaka TS | title = Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions | journal = PLOS ONE | volume = 5 | issue = 12 | pages = e15655 | date = December 2010 | pmid = 21179449 | pmc = 3001487 | doi = 10.1371/journal.pone.0015655 | veditors = Zhou Z | doi-access = free | bibcode = 2010PLoSO...515655C }}
• Lineage specification{{cite journal | vauthors = Engler AJ, Sen S, Sweeney HL, Discher DE | title = Matrix elasticity directs stem cell lineage specification | journal = Cell | volume = 126 | issue = 4 | pages = 677–689 | date = August 2006 | pmid = 16923388 | doi = 10.1016/j.cell.2006.06.044 | doi-access = free }}
• Cancer cell phenotype{{cite journal | vauthors = Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, Reinhart-King CA, Margulies SS, Dembo M, Boettiger D, Hammer DA, Weaver VM | display-authors = 6 | title = Tensional homeostasis and the malignant phenotype | journal = Cancer Cell | volume = 8 | issue = 3 | pages = 241–254 | date = September 2005 | pmid = 16169468 | doi = 10.1016/j.ccr.2005.08.010 | doi-access = free }}{{cite journal | vauthors = Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM | display-authors = 6 | title = Matrix crosslinking forces tumor progression by enhancing integrin signaling | journal = Cell | volume = 139 | issue = 5 | pages = 891–906 | date = November 2009 | pmid = 19931152 | pmc = 2788004 | doi = 10.1016/j.cell.2009.10.027 }}{{cite journal | vauthors = Tilghman RW, Cowan CR, Mih JD, Koryakina Y, Gioeli D, Slack-Davis JK, Blackman BR, Tschumperlin DJ, Parsons JT | display-authors = 6 | title = Matrix rigidity regulates cancer cell growth and cellular phenotype | journal = PLOS ONE | volume = 5 | issue = 9 | pages = e12905 | date = September 2010 | pmid = 20886123 | pmc = 2944843 | doi = 10.1371/journal.pone.0012905 | veditors = Hotchin NA | doi-access = free | bibcode = 2010PLoSO...512905T }}
• Fibrosis{{cite journal | vauthors = Liu F, Mih JD, Shea BS, Kho AT, Sharif AS, Tager AM, Tschumperlin DJ | title = Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression | journal = The Journal of Cell Biology | volume = 190 | issue = 4 | pages = 693–706 | date = August 2010 | pmid = 20733059 | pmc = 2928007 | doi = 10.1083/jcb.201004082 }}{{cite journal | vauthors = Wipff PJ, Rifkin DB, Meister JJ, Hinz B | title = Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix | journal = The Journal of Cell Biology | volume = 179 | issue = 6 | pages = 1311–1323 | date = December 2007 | pmid = 18086923 | pmc = 2140013 | doi = 10.1083/jcb.200704042 }}
• Hepatocyte function{{cite journal | vauthors = Georges PC, Hui JJ, Gombos Z, McCormick ME, Wang AY, Uemura M, Mick R, Janmey PA, Furth EE, Wells RG | display-authors = 6 | title = Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis | journal = American Journal of Physiology. Gastrointestinal and Liver Physiology | volume = 293 | issue = 6 | pages = G1147–G1154 | date = December 2007 | pmid = 17932231 | doi = 10.1152/ajpgi.00032.2007 | s2cid = 201357 }}{{cite journal | vauthors = Li L, Sharma N, Chippada U, Jiang X, Schloss R, Yarmush ML, Langrana NA | title = Functional modulation of ES-derived hepatocyte lineage cells via substrate compliance alteration | journal = Annals of Biomedical Engineering | volume = 36 | issue = 5 | pages = 865–876 | date = May 2008 | pmid = 18266108 | doi = 10.1007/s10439-008-9458-3 | s2cid = 21773886 }}{{cite journal | vauthors = Semler EJ, Lancin PA, Dasgupta A, Moghe PV | title = Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance | journal = Biotechnology and Bioengineering | volume = 89 | issue = 3 | pages = 296–307 | date = February 2005 | pmid = 15744840 | doi = 10.1002/bit.20328 }}
• Mechanosensing{{cite journal | vauthors = Friedland JC, Lee MH, Boettiger D | title = Mechanically activated integrin switch controls alpha5beta1 function | journal = Science | volume = 323 | issue = 5914 | pages = 642–644 | date = January 2009 | pmid = 19179533 | doi = 10.1126/science.1168441 | s2cid = 206517419 | bibcode = 2009Sci...323..642F }}{{cite journal | vauthors = Chan CE, Odde DJ | title = Traction dynamics of filopodia on compliant substrates | journal = Science | volume = 322 | issue = 5908 | pages = 1687–1691 | date = December 2008 | pmid = 19074349 | doi = 10.1126/science.1163595 | s2cid = 28568350 | bibcode = 2008Sci...322.1687C }}{{cite journal | vauthors = Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S | display-authors = 6 | title = Role of YAP/TAZ in mechanotransduction | journal = Nature | volume = 474 | issue = 7350 | pages = 179–183 | date = June 2011 | pmid = 21654799 | doi = 10.1038/nature10137 | hdl-access = free | s2cid = 205225137 | hdl = 11380/673649 }}

Cell culture in three dimensions has been touted as "Biology's New Dimension".{{cite web|url=http://www.nature.com/drugdisc/news/articles/424870a.html |title=drug discovery@nature.com |publisher=Nature.com |access-date=2013-03-26}} At present, the practice of cell culture remains based on varying combinations of single or multiple cell structures in 2D.{{cite journal | vauthors = Duell BL, Cripps AW, Schembri MA, Ulett GC | title = Epithelial cell coculture models for studying infectious diseases: benefits and limitations | journal = Journal of Biomedicine & Biotechnology | volume = 2011 | pages = 852419 | year = 2011 | pmid = 22007147 | pmc = 3189631 | doi = 10.1155/2011/852419 | doi-access = free }} Currently, there is an increase in use of 3D cell cultures in research areas including drug discovery, cancer biology, regenerative medicine, nanomaterials assessment and basic life science research.{{cite journal | vauthors = Barrila J, Radtke AL, Crabbé A, Sarker SF, Herbst-Kralovetz MM, Ott CM, Nickerson CA | title = Organotypic 3D cell culture models: using the rotating wall vessel to study host-pathogen interactions | journal = Nature Reviews. Microbiology | volume = 8 | issue = 11 | pages = 791–801 | date = November 2010 | pmid = 20948552 | doi = 10.1038/nrmicro2423 | s2cid = 6925183 | doi-access = free }}{{Cite journal| vauthors = Mapanao AK, Voliani V |date=June 2020|title=Three-dimensional tumor models: Promoting breakthroughs in nanotheranostics translational research|journal=Applied Materials Today|language=en|volume=19|pages=100552|doi=10.1016/j.apmt.2019.100552|s2cid=213634060}}{{Cite journal| vauthors = Cassano D, Santi M, D'Autilia F, Mapanao AK, Luin S, Voliani V |date=2019|title=Photothermal effect by NIR-responsive excretable ultrasmall-in-nano architectures|journal=Materials Horizons|language=en|volume=6|issue=3|pages=531–537|doi=10.1039/C9MH00096H|issn=2051-6347|doi-access=free|hdl=11384/77439|hdl-access=free}} 3D cell cultures can be grown using a scaffold or matrix, or in a scaffold-free manner. Scaffold based cultures utilize an acellular 3D matrix or a liquid matrix. Scaffold-free methods are normally generated in suspensions.{{cite journal | vauthors = Edmondson R, Broglie JJ, Adcock AF, Yang L | title = Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors | journal = Assay and Drug Development Technologies | volume = 12 | issue = 4 | pages = 207–218 | date = May 2014 | pmid = 24831787 | pmc = 4026212 | doi = 10.1089/adt.2014.573 }} There are a variety of platforms used to facilitate the growth of three-dimensional cellular structures including scaffold systems such as hydrogel matrices{{cite journal | vauthors = Bhattacharya M, Malinen MM, Lauren P, Lou YR, Kuisma SW, Kanninen L, Lille M, Corlu A, GuGuen-Guillouzo C, Ikkala O, Laukkanen A, Urtti A, Yliperttula M | display-authors = 6 | title = Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture | journal = Journal of Controlled Release | volume = 164 | issue = 3 | pages = 291–298 | date = December 2012 | pmid = 22776290 | doi = 10.1016/j.jconrel.2012.06.039 | doi-access = free }} and solid scaffolds, and scaffold-free systems such as low-adhesion plates, nanoparticle facilitated magnetic levitation,{{cite journal | vauthors = DeRosa MC, Monreal C, Schnitzer M, Walsh R, Sultan Y | title = Nanotechnology in fertilizers | journal = Nature Nanotechnology | volume = 5 | issue = 2 | pages = 91 | date = February 2010 | pmid = 20130583 | doi = 10.1038/nnano.2010.2 | doi-access = free | bibcode = 2010NatNa...5...91D }} hanging drop plates,{{cite journal | vauthors = Hsiao AY, Tung YC, Qu X, Patel LR, Pienta KJ, Takayama S | title = 384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids | journal = Biotechnology and Bioengineering | volume = 109 | issue = 5 | pages = 1293–1304 | date = May 2012 | pmid = 22161651 | pmc = 3306496 | doi = 10.1002/bit.24399 }}{{cite journal | vauthors = Mapanao AK, Santi M, Faraci P, Cappello V, Cassano D, Voliani V | title = Endogenously Triggerable Ultrasmall-in-Nano Architectures: Targeting Assessment on 3D Pancreatic Carcinoma Spheroids | journal = ACS Omega | volume = 3 | issue = 9 | pages = 11796–11801 | date = September 2018 | pmid = 30320273 | pmc = 6173554 | doi = 10.1021/acsomega.8b01719 }} and rotary cell culture. Culturing cells in 3D leads to wide variation in gene expression signatures and partly mimics tissues in the physiological states.{{cite journal | vauthors = Ghosh S, Börsch A, Ghosh S, Zavolan M | title = The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins | journal = BMC Genomics | volume = 22 | issue = 1 | pages = 238 | date = April 2021 | pmid = 33823809 | pmc = 8025518 | doi = 10.1186/s12864-021-07532-2 | doi-access = free }} A 3D cell culture model showed cell growth similar to that of in vivo than did a monolayer culture, and all three cultures were capable of sustaining cell growth.{{cite journal | vauthors = Fontoura JC, Viezzer C, Dos Santos FG, Ligabue RA, Weinlich R, Puga RD, Antonow D, Severino P, Bonorino C | display-authors = 6 | title = Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance | journal = Materials Science & Engineering. C, Materials for Biological Applications | volume = 107 | pages = 110264 | date = February 2020 | pmid = 31761183 | doi = 10.1016/j.msec.2019.110264 | hdl = 10923/20413 | s2cid = 208277016 | hdl-access = free }} As 3D culturing has been developed it turns out to have a great potential to design tumors models and investigate malignant transformation and metastasis, 3D cultures can provide aggerate tool for understanding changes, interactions, and cellular signaling.{{cite journal | vauthors = Habanjar O, Diab-Assaf M, Caldefie-Chezet F, Delort L | title = 3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages | journal = International Journal of Molecular Sciences | volume = 22 | issue = 22 | pages = 12200 | date = November 2021 | pmid = 34830082 | pmc = 8618305 | doi = 10.3390/ijms222212200 | doi-access = free }}

Eric Simon, in a 1988 NIH SBIR grant report, showed that electrospinning could be used to produce nano- and submicron-scale polystyrene and polycarbonate fibrous scaffolds specifically intended for use as in vitro cell substrates. This early use of electrospun fibrous lattices for cell culture and tissue engineering showed that various cell types including Human Foreskin Fibroblasts (HFF), transformed Human Carcinoma (HEp-2), and Mink Lung Epithelium (MLE) would adhere to and proliferate upon polycarbonate fibers. It was noted that, as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more histotypic rounded 3-dimensional morphology generally observed in vivo.

As the natural extracellular matrix (ECM) is important in the survival, proliferation, differentiation and migration of cells, different hydrogel culture matrices mimicking natural ECM structure are seen as potential approaches to in vivo–like cell culturing.{{cite journal | vauthors = Tibbitt MW, Anseth KS | title = Hydrogels as extracellular matrix mimics for 3D cell culture | journal = Biotechnology and Bioengineering | volume = 103 | issue = 4 | pages = 655–663 | date = July 2009 | pmid = 19472329 | pmc = 2997742 | doi = 10.1002/bit.22361 }} Hydrogels are composed of interconnected pores with high water retention, which enables efficient transport of substances such as nutrients and gases. Several different types of hydrogels from natural and synthetic materials are available for 3D cell culture, including animal ECM extract hydrogels, protein hydrogels, peptide hydrogels, polymer hydrogels, and wood-based nanocellulose hydrogel.

The 3D Cell Culturing by Magnetic Levitation method (MLM) is the application of growing 3D tissue by inducing cells treated with magnetic nanoparticle assemblies in spatially varying magnetic fields using neodymium magnetic drivers and promoting cell to cell interactions by levitating the cells up to the air/liquid interface of a standard petri dish. The magnetic nanoparticle assemblies consist of magnetic iron oxide nanoparticles, gold nanoparticles, and the polymer polylysine. 3D cell culturing is scalable, with the capability for culturing 500 cells to millions of cells or from single dish to high-throughput low volume systems.

Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells in vitro.

The major application of human cell culture is in stem cell industry, where mesenchymal stem cells can be cultured and cryopreserved for future use. Tissue engineering potentially offers dramatic improvements in low cost medical care for hundreds of thousands of patients annually.

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,{{cite magazine|url=https://www.wired.com/news/wireservice/0,70102-0.html?tw=wn_index_7 |title=Quickie Bird Flu Vaccine Created|date=2006-01-26|agency=Reuters|magazine=Wired|access-date=2010-01-31}}{{cite journal | vauthors = 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 | display-authors = 6 | 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 | date = February 2006 | pmid = 16439551 | pmc = 1367171 | doi = 10.1128/JVI.80.4.1959-1964.2006 }}

and novel adjuvants.{{cite web|url=https://www.niaid.nih.gov/news/newsreleases/2004/pages/h9n2.aspx|title=NIAID Taps Chiron to Develop Vaccine Against H9N2 Avian Influenza|date=2004-08-17|work=National Institute of Allergy and Infectious Diseases (NIAID)|access-date=2010-01-31}}

The technique of co-culturing is used to study cell crosstalk between two or more types of cells on a plate or in a 3D matrix. The cultivation of different stem cells and the interaction of immune cells can be investigated in an in vitro model similar to biological tissue. Since most tissues contain more than one type of cell, it is important to evaluate their interaction in a 3D culture environment to gain a better understanding of their interaction and to introduce mimetic tissues. There are two types of co-culturing: direct and indirect. While direct interaction involves cells being in direct contact with each other in the same culture media or matrix, indirect interaction involves different environments, allowing signaling and soluble factors to participate.{{Cite journal |last1=Miki |first1=Yasuhiro |last2=Ono |first2=Katsuhiko |last3=Hata |first3=Shuko |last4=Suzuki |first4=Takashi |last5=Kumamoto |first5=Hiroyuki |last6=Sasano |first6=Hironobu |date=September 2012 |title=The advantages of co-culture over mono cell culture in simulating in vivo environment |url=http://dx.doi.org/10.1016/j.jsbmb.2011.12.004 |journal=The Journal of Steroid Biochemistry and Molecular Biology |volume=131 |issue=3–5 |pages=68–75 |doi=10.1016/j.jsbmb.2011.12.004 |pmid=22265957 |s2cid=19646957 |issn=0960-0760}}

Cell differentiation in tissue models during interaction between cells can be studied using the Co-Cultured System to simulate cancer tumors, to assess the effect of drugs on therapeutic trials, and to study the effect of drugs on therapeutic trials. The co-culture system in 3D models can predict the response to chemotherapy and endocrine therapy if the microenvironment defines biological tissue for the cells.

A co-culture method is used in tissue engineering to generate tissue formation with multiple cells interacting directly.{{Cite journal |last1=Paschos |first1=Nikolaos K. |last2=Brown |first2=Wendy E. |last3=Eswaramoorthy |first3=Rajalakshmanan |last4=Hu |first4=Jerry C. |last5=Athanasiou |first5=Kyriacos A. |date=2014-02-03 |title=Advances in tissue engineering through stem cell-based co-culture |journal=Journal of Tissue Engineering and Regenerative Medicine |volume=9 |issue=5 |pages=488–503 |doi=10.1002/term.1870 |pmid=24493315 |s2cid=1991776 |issn=1932-6254|doi-access=free }}

File:Cell culture-fig.png

{{Main article|Microfluidic cell culture}}Microfluidics technique is developed systems that can perform a process in a flow which are usually in a scale of micron. Microfluidics chip are also known as Lab-on-a-chip and they are able to have continuous procedure and reaction steps with spare amount of reactants and space. Such systems enable the identification and isolation of individual cells and molecules when combined with appropriate biological assays and high-sensitivity detection techniques.{{Cite journal |last1=Dittrich |first1=Petra S. |last2=Manz |first2=Andreas |date=March 2006 |title=Lab-on-a-chip: microfluidics in drug discovery |url=https://www.nature.com/articles/nrd1985 |journal=Nature Reviews Drug Discovery |language=en |volume=5 |issue=3 |pages=210–218 |doi=10.1038/nrd1985 |pmid=16518374 |s2cid=35904402 |issn=1474-1784}}{{Cite journal |last1=Terrell |first1=John A. |last2=Jones |first2=Curtis G. |last3=Kabandana |first3=Giraso Keza Monia |last4=Chen |first4=Chengpeng |date=2020 |title=From cells-on-a-chip to organs-on-a-chip: scaffolding materials for 3D cell culture in microfluidics |url=https://pubs.rsc.org/en/content/articlelanding/2020/tb/d0tb00718h |journal=Journal of Materials Chemistry B |language=en |volume=8 |issue=31 |pages=6667–6685 |doi=10.1039/D0TB00718H|pmid=32567628 |hdl=11603/21825 |s2cid=219972841 |hdl-access=free }}

{{Main|Organ-on-a-chip}}

OoC systems mimic and control the microenvironment of the cells by growing tissues in microfluidics. Combining tissue engineering, biomaterials fabrication, and cell biology, it offers the possibility of establishing a biomimetic model for studying human diseases in the laboratory. In recent years, 3D cell culture science has made significant progress, leading to the development of OoC. OoC is considered as a preclinical step that benefits pharmaceutical studies, drug development and disease modeling.{{Cite journal |last1=Wu |first1=Qirui |last2=Liu |first2=Jinfeng |last3=Wang |first3=Xiaohong |last4=Feng |first4=Lingyan |last5=Wu |first5=Jinbo |last6=Zhu |first6=Xiaoli |last7=Wen |first7=Weijia |last8=Gong |first8=Xiuqing |date=2020-02-12 |title=Organ-on-a-chip: recent breakthroughs and future prospects |journal=BioMedical Engineering OnLine |language=en |volume=19 |issue=1 |pages=9 |doi=10.1186/s12938-020-0752-0 |issn=1475-925X |pmc=7017614 |pmid=32050989 |doi-access=free }}{{Cite journal |last1=Leung |first1=Chak Ming |last2=de Haan |first2=Pim |last3=Ronaldson-Bouchard |first3=Kacey |last4=Kim |first4=Ge-Ah |last5=Ko |first5=Jihoon |last6=Rho |first6=Hoon Suk |last7=Chen |first7=Zhu |last8=Habibovic |first8=Pamela |last9=Jeon |first9=Noo Li |last10=Takayama |first10=Shuichi |last11=Shuler |first11=Michael L. |last12=Vunjak-Novakovic |first12=Gordana |last13=Frey |first13=Olivier |last14=Verpoorte |first14=Elisabeth |last15=Toh |first15=Yi-Chin |date=2022-05-12 |title=A guide to the organ-on-a-chip |journal=Nature Reviews Methods Primers |language=en |volume=2 |issue=1 |pages=1–29 |doi=10.1038/s43586-022-00118-6 |s2cid=248756548 |issn=2662-8449|doi-access=free }} OoC is an important technology that can bridge the gap between animal testing and clinical studies and also by the advances that the science has achieved could be a replace for in vivo studies for drug delivery and pathophysiological studies.{{Cite journal |last1=Ma |first1=Chao |last2=Peng |first2=Yansong |last3=Li |first3=Hongtong |last4=Chen |first4=Weiqiang |date=February 2021 |title=Organ-on-a-Chip: A New Paradigm for Drug Development |journal=Trends in Pharmacological Sciences |language=en |volume=42 |issue=2 |pages=119–133 |doi=10.1016/j.tips.2020.11.009 |pmc=7990030 |pmid=33341248}}

Besides the culture of well-established immortalised cell lines, cells from primary explants of a plethora of organisms can be cultured for a limited period of time before senescence occurs (see Hayflick's limit). Cultured primary cells have been extensively used in research, as is the case of fish keratocytes in cell migration studies.{{cite journal | vauthors = Rapanan JL, Cooper KE, Leyva KJ, Hull EE | title = Collective cell migration of primary zebrafish keratocytes | journal = Experimental Cell Research | volume = 326 | issue = 1 | pages = 155–165 | date = August 2014 | pmid = 24973510 | doi = 10.1016/j.yexcr.2014.06.011 }}{{cite journal | vauthors = Lee J, Jacobson K | title = The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes | journal = Journal of Cell Science | volume = 110 | issue = 22 | pages = 2833–2844 | date = November 1997 | pmid = 9427291 | doi = 10.1242/jcs.110.22.2833 | url = https://cdr.lib.unc.edu/downloads/xw42nh90z }}

{{main|Plant tissue culture}}

{{see also|Tobacco BY-2 cells}}

Plant cell cultures are typically grown as cell suspension cultures in a liquid medium or as callus cultures on a solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin.{{cn|date=December 2023}}

{{main|Insect cell culture}}

Cells derived from Drosophila melanogaster (most prominently, Schneider 2 cells) can be used for experiments which may be hard to do on live flies or larvae, such as biochemical studies or studies using siRNA. Cell lines derived from the army worm Spodoptera frugiperda, including Sf9 and Sf21, and from the cabbage looper Trichoplusia ni, High Five cells, are commonly used for expression of recombinant proteins using baculovirus.{{cite journal | vauthors = Drugmand JC, Schneider YJ, Agathos SN | title = Insect cells as factories for biomanufacturing | journal = Biotechnology Advances | volume = 30 | issue = 5 | pages = 1140–1157 | date = 2012 | pmid = 21983546 | doi = 10.1016/j.biotechadv.2011.09.014 | url = https://zenodo.org/record/896333 }}

{{main|Microbiological culture}}

For bacteria and yeasts, 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.{{cn|date=December 2023}}

{{main|Viral culture}}

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. 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.{{cn|date=December 2023}}

;Human cell lines

• DU145 (prostate cancer)
• H295R (adrenocortical cancer)
• HeLa (cervical cancer)
• KBM-7 (chronic myelogenous leukemia)
• LNCaP (prostate cancer)
• MCF-7 (breast cancer)
• MDA-MB-468 (breast cancer)
• PC3 (prostate cancer)
• SaOS-2 (bone cancer)
• SH-SY5Y (neuroblastoma, cloned from a myeloma)
• T-47D (breast cancer)
• THP-1 (acute myeloid leukemia)
• U-87 MG (glioblastoma)
• National Cancer Institute's 60 cancer cell line panel (NCI60)

;Animal cell lines

• Vero (African green monkey Chlorocebus kidney epithelial cell line)
• BHK21 cell (Baby Hambster Kidney)
• MDBK cell (Madin-Darby Bovine Kidney)
• DF-1cell (chicken fibroblast)

;Mouse cell lines

• MC3T3 (embryonic calvarium)

;Rat tumor cell lines

• GH3 (pituitary tumor)
• PC12 (pheochromocytoma)

;Plant cell lines

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

;Other species cell lines

• Dog MDCK kidney epithelial
• Xenopus A6 kidney epithelial
• Zebrafish AB9

{{Incomplete list|date=July 2011}}

• Biological immortality
• Cell culture assays
• Electric cell-substrate impedance sensing
• List of contaminated cell lines
• List of NCI-60 Cell Lines
• List of LL-100 panel Cell Lines
• List of breast cancer cell lines
• Microphysiometry

{{reflist|30em}}

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• {{cite journal |doi=10.1038/nprot.2006.294 |title=Rat Chromaffin cells primary cultures: Standardization and quality assessment for single-cell assays |year=2006 | vauthors = Gilabert JA, Montalvo GB, Artalejo AR |journal=Protocol Exchange|doi-access=free }}
• {{cite journal |doi=10.4067/S0717-95022015000200059 |title=Sergey Fedoroff: A Pioneer of the Neuronal Regeneration. Tribute from the Pan American Association of Anatomy |year=2015 | vauthors = Losardo RJ, Gutiérrez RC, Prates JC, Moscovici M, Torres AR, Martínez MA |journal= International Journal of Morphology|volume=33 |issue=2 |pages=794–800 |doi-access=free }}
• {{cite journal | vauthors = MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG | title = Widespread intraspecies cross-contamination of human tumor cell lines arising at source | journal = International Journal of Cancer | volume = 83 | issue = 4 | pages = 555–563 | date = November 1999 | pmid = 10508494 | doi = 10.1002/(SICI)1097-0215(19991112)83:4<555::AID-IJC19>3.0.CO;2-2 | doi-access = free }}
• {{cite journal | vauthors = Masters JR | title = HeLa cells 50 years on: the good, the bad and the ugly | journal = Nature Reviews. Cancer | volume = 2 | issue = 4 | pages = 315–319 | date = April 2002 | pmid = 12001993 | doi = 10.1038/nrc775 | s2cid = 991019 }}
• {{cite journal | vauthors = Witkowski JA | title = Experimental pathology and the origins of tissue culture: Leo Loeb's contribution | journal = Medical History | volume = 27 | issue = 3 | pages = 269–288 | date = July 1983 | pmid = 6353093 | pmc = 1139336 | doi = 10.1017/S0025727300042964 }}

{{refend}}

{{Library resources box

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• [https://www.ncbi.nlm.nih.gov/books/NBK26851/table/A1515/ Table of common cell lines from Alberts 4th ed.]
• [https://web.archive.org/web/20090708143546/http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CancerCellsInCulture.html Cancer Cells in Culture]
• [https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/evolution-of-cell Evolution of Cell Culture Surfaces]
• [https://web.archive.org/web/20100116184727/http://bioinformatics.istge.it/hypercldb/ Hypertext version of the Cell Line Data Base]
• [http://www.corning.com/worldwide/en/products/life-sciences/applications/cell-culture.html Cell Culture Applications] - Resources including application notes and protocols to create an ideal environment for growing cells, right from the start.
• [https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html Cell Culture Basics] - Introduction to cell culture, covering topics such as laboratory set-up, safety and aseptic technique including basic cell culture protocols and video training
• [https://web.archive.org/web/20151121002905/http://www.mavensemantic.com/ Database of Who's Who in Cell Culture and Related Research]
• [https://web.archive.org/web/20030830095140/http://ccr.coriell.org/ Coriell Cell Repositories]
• [http://www.corning.com/worldwide/en/products/life-sciences/resources/webinars.html?commid=153597 An Introduction To Cell Culture.] This webinar introduces the history, theory, basic techniques, and potential pit-falls of mammalian cell culture.
• [https://nccs.res.in/ The National Centre for Cell Science] (NCCS), Pune, India; national repository for cell lines/hybridomas etc.
• [https://www.culturecollections.org.uk/collections/ecacc.aspx Public Health England] {{Webarchive|url=https://web.archive.org/web/20220607095449/https://www.culturecollections.org.uk/collections/ecacc.aspx |date=7 June 2022 }}, Public Health England Culture Collections (ECACC)

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{{Molecular biology}}

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{{DEFAULTSORT:Cell Culture}}

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