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Cell culture or tissue culture is the process by which
cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This technique is also called micropropagation. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions the need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients ( amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases ( CO2, O2), and regulates the physio-chemical environment ( pH buffer,
osmotic pressure Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward flow of its pure solvent across a semipermeable membrane. It is also defined as the measure of the tendency of a solution to take in a pure ...
, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid
growth medium A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...
, such as broth or
agar Agar ( or ), or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from ogonori (''Gracilaria'') and "tengusa" (''Gelidiaceae''). As found in nature, agar is ...
. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The lifespan of most cells is genetically determined, but some cell culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided. In practice, the term "cell culture" now refers to the culturing of cells derived from multicellular
eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
s, especially animal cells, in contrast with other types of culture that also grow cells, such as plant tissue culture, fungal culture, and
microbiological culture A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagn ...
(of
microbe A microorganism, or microbe,, ''mikros'', "small") and ''organism'' from the el, ὀργανισμός, ''organismós'', "organism"). It is usually written as a single word but is sometimes hyphenated (''micro-organism''), especially in olde ...
s). The historical development and methods of cell culture are closely interrelated to those of tissue culture and
organ culture Organ culture is a development from tissue culture methods of research, the organ culture is able to accurately model functions of an organ in various states and conditions by the use of the actual ''in vitro'' organ itself. Parts of an organ or ...
. Viral culture is also related, with cells as hosts for the viruses. The laboratory technique of maintaining live cell lines (a population of cells descended from a single cell and containing the same genetic makeup) separated from their original tissue source became more robust in the middle 20th century.


History

The 19th-century English physiologist
Sydney Ringer Sydney Ringer FRS (March 1835 – 14 October 1910) was a British clinician, physiologist and pharmacologist, best known for inventing Ringer's solution. He was born in 1835 in Norwich, England and died following a stroke in 1910 in Lastingham, Y ...
developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated
animal heart ''Animal Heart'' is the debut studio album by Nina Persson, singer of The Cardigans, released on February 10, 2014 through Lojinx in Europe and The End Records in North America. A video for the title track of the album was released in November ...
outside the body. In 1885 Wilhelm Roux removed a section of the
medullary plate The neural plate is a key developmental structure that serves as the basis for the nervous system. Cranial to the primitive node of the embryonic primitive streak, ectodermal tissue thickens and flattens to become the neural plate. The region ante ...
of an
embryo An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...
nic 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. 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. 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. Gottlieb Haberlandt first pointed out the possibilities of the culture of isolated tissues, plant tissue culture. 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. His original idea, presented in 1902, was called totipotentiality: “Theoretically all plant cells are able to give rise to a complete plant.” 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 Thomas Huckle Weller (June 15, 1915 – August 23, 2008) was an American virologist. He, John Franklin Enders and Frederick Chapman Robbins were awarded a Nobel Prize in Physiology or Medicine in 1954 for showing how to cultivate poliomyelitis ...
, 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.


Modern usage

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 An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
. The cells are bathed in a culture medium, which contains essential nutrients and energy sources necessary for the cells' survival. 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). Eric Simon, in a 1988 NIH SBIR grant report, showed that electrospinning could be used to produced 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''. Plant tissue culture in particular is concerned with the growing of entire plants from small pieces of plant tissue, cultured in medium.


Concepts in mammalian cell culture


Isolation of cells

Cells can be
isolated Isolation is the near or complete lack of social contact by an individual. Isolation or isolated may also refer to: Sociology and psychology *Isolation (health care), various measures taken to prevent contagious diseases from being spread **Is ...
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. 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.


Maintaining cells in culture

For the majority of isolated primary cells, they undergo the process of
senescence Senescence () or biological aging is the gradual deterioration of functional characteristics in living organisms. The word ''senescence'' can refer to either cellular senescence or to senescence of the whole organism. Organismal senescence inv ...
and stop dividing after a certain number of population doublings while generally retaining their viability (described as the Hayflick limit). Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the cell
growth medium A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...
. 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
prion Prions are misfolded proteins that have the ability to transmit their misfolded shape onto normal variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in humans and many other animals. It ...
s, particularly in medical biotechnology applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible and use human
platelet lysate Human platelet lysate (or hPL) is a substitute supplement for fetal bovine serum (FBS) in experimental and clinical cell culture. It is a turbid, light-yellow liquid that is obtained from human blood platelets after freeze/thaw cycle(s). The freeze/ ...
(hPL). 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, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture. 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 Progesterone (P4) is an endogenous steroid and progestogen sex hormone involved in the menstrual cycle, pregnancy, and embryogenesis of humans and other species. It belongs to a group of steroid hormones called the progestogens and is the m ...
-producing
theca lutein cell The corpus luteum (Latin for "yellow body"; plural corpora lutea) is a temporary endocrine structure in female ovaries involved in the production of relatively high levels of progesterone, and moderate levels of estradiol, and inhibin A. It is t ...
s. Cells can be grown either 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 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 A microcarrier is a support matrix that allows for the growth of adherent cells in bioreactors. Instead of on a flat surface, cells are cultured on the surface of spherical microcarriers so that each particle carries several hundred cells, and the ...
, 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).


Cell culture basal media

There are different kinds of cell culture media which being used routinely in life science including the following: *
MEM Mem (also spelled Meem, Meme, or Mim) is the thirteenth letter of the Semitic abjads, including Hebrew mēm , Aramaic Mem , Syriac mīm ܡ, Arabic mīm and Phoenician mēm . Its sound value is . The Phoenician letter gave rise to the Greek mu ...
* DMEM *
RPMI 1640 RPMI 1640, also known as RPMI medium, is a growth medium used in cell culture. RPMI 1640 was developed by George E. Moore, Robert E. Gerner, and H. Addison Franklin in 1966 at Roswell Park Comprehensive Cancer Center (formerly known as Roswell Park ...
* Ham's f-12 * IMDM *Leibovitz L-15 *DMEM/F-12


Components of cell culture media


Typical Growth conditions


Cell line cross-contamination

Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest anywhere from 15 to 20% of the time, cells used in experiments have been misidentified or contaminated with another cell line. Problems with cell line cross-contamination have even been detected in lines from the
NCI-60 panel The NCI-60 cancer cell line panel is a group of 60 human cancer cell lines used by the National Cancer Institute (NCI) for the screening of compounds to detect potential anticancer activity. Purpose The screening procedure is called the NCI-60 ...
, which are used routinely for drug-screening studies. 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. Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions. ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines. To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. Many methods are used to identify cell lines, including isoenzyme analysis,
human lymphocyte antigen The human leukocyte antigen (HLA) system or complex is a complex of genes on chromosome 6 in humans which encode cell-surface proteins responsible for the regulation of the immune system. The HLA system is also known as the human version of th ...
(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.


Other technical issues

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 Cellular differentiation is the process in which a stem cell alters from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular ...
. * Genetic and
epigenetic In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
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. 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. A systematic bias in generated datasets was recently shown for
CRISPR CRISPR () (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacte ...
and RNAi gene silencing screens, and for metabolic profiling of cancer cell lines. Using a
growth medium A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...
that better represents the physiological levels of nutrients can improve the physiological relevance of in vitro studies and recently such media types, as Plasmax and Human Plasma Like Medium (HPLM), were developed.


Manipulation of cultured cells

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 Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
and streptomycin) and antifungals (e.g. amphotericin B and
Antibiotic-Antimycotic Antibiotic-Antimycotic is a solution that is commonly added to cell culture media to prevent contamination by various bacteria and fungi. It generally contains 10,000 units per milliliter penicillin, 10,000 micrograms per milliliter streptomycin ...
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.


Media changes

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.


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. Some cell cultures, such as
RAW cells Raw is an adjective usually describing: * Raw materials, basic materials from which products are manufactured or made * Raw food, uncooked food Raw or RAW may also refer to: Computing and electronics * .RAW, a proprietary mass spectrometry data ...
are mechanically scraped from the surface of their vessel with rubber scrapers.


Transfection and transduction

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.


Established human cell lines

Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the
Supreme Court of California The Supreme Court of California is the highest and final court of appeals in the courts of the U.S. state of California. It is headquartered in San Francisco at the Earl Warren Building, but it regularly holds sessions in Los Angeles and Sacra ...
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. It is possible to fuse normal cells with an
immortalised cell line An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
. This method is used to produce
monoclonal antibodies A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Monoclonal antibodies ca ...
. In brief, lymphocytes isolated from the spleen (or possibly blood) of an
immunised Immunization, or immunisation, is the process by which an individual's immune system becomes fortified against an infectious agent (known as the antigen, immunogen). When this system is exposed to molecules that are foreign to the body, called ...
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 A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differe ...
(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.


Cell strains

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 and, after this, they lose their ability to proliferate (a genetically determined event known as senescence).


Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology. Culture of human
stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...
s is used to expand the number of cells and differentiate the cells into various somatic cell types for transplantation. Stem cell culture is also used to harvest the molecules and exosomes that the stem cells release for the purposes of therapeutic development. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include
enzymes Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
, synthetic
hormones A hormone (from the Ancient Greek, Greek participle , "setting in motion") is a class of cell signaling, signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and beh ...
, immunobiologicals (
monoclonal antibodies A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a cell Lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Monoclonal antibodies ca ...
, interleukins, lymphokines), and
anticancer agents Chemotherapy (often abbreviated to chemo and sometimes CTX or CTx) is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents or alkylating agents) as part of a standardized chemotherapy regimen. Chemotherap ...
. 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 Erythropoietin (; EPO), also known as erythropoetin, haematopoietin, or haemopoietin, is a glycoprotein cytokine secreted mainly by the kidneys in response to cellular hypoxia; it stimulates red blood cell production (erythropoiesis) in the bo ...
. 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 Somatic may refer to: * Somatic (biology), referring to the cells of the body in contrast to the germ line cells ** Somatic cell, a non-gametic cell in a multicellular organism * Somatic nervous system, the portion of the vertebrate nervous sys ...
embryos as a source for direct gene transfer via particle bombardment, transit
gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
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.


Cell culture in two dimensions

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 A Petri dish (alternatively known as a Petri plate or cell-culture dish) is a shallow transparent lidded dish that biologists use to hold growth medium in which cells can be cultured,R. C. Dubey (2014): ''A Textbook Of Biotechnology For Class- ...
. Julius Richard Petri, a German bacteriologist, is generally credited with this invention while working as an assistant to
Robert Koch Heinrich Hermann Robert Koch ( , ; 11 December 1843 – 27 May 1910) was a German physician and microbiologist. As the discoverer of the specific causative agents of deadly infectious diseases including tuberculosis, cholera (though the Vibrio ...
. 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 Matrix most commonly refers to: * ''The Matrix'' (franchise), an American media franchise ** ''The Matrix'', a 1999 science-fiction action film ** "The Matrix", a fictional setting, a virtual reality environment, within ''The Matrix'' (franchis ...
, a concept that has led to discoveries in fields such as: *Stem cell self-renewal *Lineage specification *Cancer cell phenotype *Fibrosis *Hepatocyte function *Mechanosensing


Cell culture in three dimensions

Cell culture in three dimensions has been touted as "Biology's New Dimension". At present, the practice of cell culture remains based on varying combinations of single or multiple cell structures in 2D. Currently, there is an increase in use of 3D cell cultures in research areas including
drug discovery In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which new candidate medications are discovered. Historically, drugs were discovered by identifying the active ingredient from traditional remedies or by ...
, cancer biology, regenerative medicine, nanomaterials assessment and basic life science research. 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. There are a variety of platforms used to facilitate the growth of three-dimensional cellular structures including scaffold systems such as hydrogel matrices and solid scaffolds, and scaffold-free systems such as low-adhesion plates, nanoparticle facilitated magnetic levitation, and hanging drop plates. Culturing cells in 3D leads to wide variation in gene expression signatures and partly mimics tissues in the physiological states. 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. 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. 3D cell culture in scaffolds Eric Simon, in a 1988 NIH SBIR grant report, showed that electrospinning could be used to produced 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''.


3D cell culture in hydrogels

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. 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.


3D Cell Culturing by Magnetic Levitation

The
3D Cell Culturing by Magnetic Levitation 3-D, 3D, or 3d may refer to: Science, technology, and mathematics Relating to three-dimensionality * Three-dimensional space ** 3D computer graphics, computer graphics that use a three-dimensional representation of geometric data ** 3D film, a ...
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.


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 ''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

Vaccines for polio,
measles Measles is a highly contagious infectious disease caused by measles virus. Symptoms usually develop 10–12 days after exposure to an infected person and last 7–10 days. Initial symptoms typically include fever, often greater than , cough, ...
,
mumps MUMPS ("Massachusetts General Hospital Utility Multi-Programming System"), or M, is an imperative, high-level programming language with an integrated transaction processing key–value database. It was originally developed at Massachusetts Gener ...
,
rubella Rubella, also known as German measles or three-day measles, is an infection caused by the rubella virus. This disease is often mild, with half of people not realizing that they are infected. A rash may start around two weeks after exposure and ...
, and
chickenpox Chickenpox, also known as varicella, is a highly contagious disease caused by the initial infection with varicella zoster virus (VZV). The disease results in a characteristic skin rash that forms small, itchy blisters, which eventually scab ...
are currently made in cell cultures. Due to the H5N1
pandemic A pandemic () is an epidemic of an infectious disease that has spread across a large region, for instance multiple continents or worldwide, affecting a substantial number of individuals. A widespread endemic (epidemiology), endemic disease wi ...
threat, research into using cell culture for
influenza vaccine Influenza vaccines, also known as flu shots, are vaccines that protect against infection by influenza viruses. New versions of the vaccines are developed twice a year, as the influenza virus rapidly changes. While their effectiveness varies fr ...
s 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 Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
(a common cold virus) as a vector, and novel adjuvants.


Cell Co-Culture

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. 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.


Cell culture in microfluidic device

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.


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. 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.


Culture of non-mammalian cells

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.


Plant cell culture methods

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 Auxins (plural of auxin ) are a class of plant hormones (or plant-growth regulators) with some morphogen-like characteristics. Auxins play a cardinal role in coordination of many growth and behavioral processes in plant life cycles and are essenti ...
and cytokinin.


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.


Bacterial and yeast culture methods

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.


Viral culture methods

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.


Common cell lines

;Human cell lines * DU145 (
prostate cancer Prostate cancer is cancer of the prostate. Prostate cancer is the second most common cancerous tumor worldwide and is the fifth leading cause of cancer-related mortality among men. The prostate is a gland in the male reproductive system that sur ...
) *
H295R H295R (also referred to as NCI-H295R) is an angiotensin-II-responsive steroid-producing adrenocortical cell line. It was initially isolated in 1980 from a 48-year-old female patient diagnosed with adrenocortical carcinoma Adrenocortical carcinoma ...
(
adrenocortical cancer Adrenocortical carcinoma (ACC) is an aggressive cancer originating in the cortex (steroid hormone-producing tissue) of the adrenal gland. Adrenocortical carcinoma is remarkable for the many hormonal syndromes that can occur in patients with ster ...
) * HeLa (
cervical cancer Cervical cancer is a cancer arising from the cervix. It is due to the abnormal growth of cells that have the ability to invade or spread to other parts of the body. Early on, typically no symptoms are seen. Later symptoms may include abnormal ...
) * KBM-7 (
chronic myelogenous leukemia Chronic myelogenous leukemia (CML), also known as chronic myeloid leukemia, is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulat ...
) * LNCaP (prostate cancer) * MCF-7 ( breast cancer) * MDA-MB-468 (breast cancer) * PC3 (prostate cancer) * SaOS-2 ( bone cancer) *
SH-SY5Y SH-SY5Y is a human derived cell line used in scientific research. The original cell line, called SK-N-SH, from which it was subcloned was isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma. SH-SY5Y cells are o ...
( neuroblastoma, cloned from a myeloma) * T-47D (breast cancer) * THP-1 (acute
myeloid Myeloid tissue, in the bone marrow sense of the word '' myeloid'' ('' myelo-'' + ''-oid''), is tissue of bone marrow, of bone marrow cell lineage, or resembling bone marrow, and myelogenous tissue (''myelo-'' + '' -genous'') is any tissue of, ...
leukemia) * U87 ( glioblastoma) * National Cancer Institute's 60 cancer cell line panel (
NCI60 The NCI-60 cancer cell line panel is a group of 60 human cancer cell lines used by the National Cancer Institute (NCI) for the screening of compounds to detect potential anticancer activity. Purpose The screening procedure is called the NCI-60 ...
) ; Primate cell lines * Vero (African green monkey ''
Chlorocebus ''Chlorocebus'' is a genus of medium-sized primates from the family of Old World monkeys. Six species are currently recognized, although some people classify them all as a single species with numerous subspecies. Either way, they make up the enti ...
'' kidney epithelial cell line) ;
Mouse A mouse ( : mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...
cell lines * MC3T3 (embryonic calvarium) ;
Rat Rats are various medium-sized, long-tailed rodents. Species of rats are found throughout the order Rodentia, but stereotypical rats are found in the genus ''Rattus''. Other rat genera include ''Neotoma'' ( pack rats), ''Bandicota'' (bandicoot ...
tumor cell lines *GH3 ( pituitary tumor) * PC12 ( pheochromocytoma) ;Plant cell lines *
Tobacco BY-2 cells {{Technical, date=September 2010 Tobacco BY-2 cells is a cell line of plant cells, which was established from a callus induced on a seedling of ''Nicotiana tabacum cv. BY-2'' (cultivar Bright Yellow - 2 of the tobacco plant). Overview Tobacco BY- ...
(kept as
cell suspension culture A cell suspension or suspension culture is a type of cell culture in which single cells or small aggregates of cells are allowed to function and multiply in an agitated growth medium, thus forming a suspension. Suspension culture is one of the two ...
, they are
model system Systems modeling or system modeling is the interdisciplinary study of the use of models to conceptualize and construct systems in business and IT development.Dog MDCK kidney epithelial * Xenopus A6 kidney epithelial * Zebrafish
AB9 Zebrafish AB9 cells are a primary fibroblast cell line developed from fin tissue of the AB strain. These cells are commonly used for studies focusing on the biochemical and molecular properties of zebrafish. Cells are grown in Dulbecco's modified ...


List of cell lines


See also

*
Biological immortality Biological immortality (sometimes referred to as bio-indefinite mortality) is a state in which the rate of mortality from senescence is stable or decreasing, thus decoupling it from chronological age. Various unicellular and multicellular species, ...
* 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 Scientists study the behaviour of isolated cells grown in the laboratory for insights into how cells function in the body in health and disease. Experiments using cell culture Cell culture or tissue culture is the process by which cells are ...
* Microphysiometry


References and notes


Further reading

* * * * * *


External links


Table of common cell lines from Alberts 4th ed.Hypertext version of the Cell Line Data BaseMicrocarrier Cell Culture Handbook by GE Healthcare Life Sciences
- Resources including application notes and protocols to create an ideal environment for growing cells, right from the start.

- Introduction to cell culture, covering topics such as laboratory set-up, safety and aseptic technique including basic cell culture protocols and video training
Database of Who's Who in Cell Culture and Related ResearchCoriell Cell RepositoriesStrategies for Protein Purification Handbook
This webinar introduces the history, theory, basic techniques, and potential pit-falls of mammalian cell culture.
The National Centre for Cell Science
(NCCS), Pune, India; national repository for cell lines/hybridomas etc.
Public Health England
Public Health England Culture Collections (ECACC) {{DEFAULTSORT:Cell Culture Biotechnology Cell biology Molecular biology techniques Alternatives to animal testing