Cell culture

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"Co-culture" redirects here. For the concept of cultures-within-cultures, see Subculture.

Cell culture in a small Petri dish
Epithelial cells in culture, stained for keratin (red) and DNA (green)

Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. After cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator.[1] 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, 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.[2] This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. 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 eukaryotes, especially animal cells, in contrast with other types of culture that also grow cells, such as plant tissue culture, fungal culture, and microbiological culture (of microbes). The historical development and methods of cell culture are closely interrelated with those of tissue culture and organ culture. 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.[3][4]

History

The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside the body.[5] 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.[6] 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.[7][8][9] 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.[10]

Gottlieb Haberlandt first pointed out the possibilities of the culture of isolated tissues, plant tissue culture.[11] 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."[12][13][14] The term tissue culture was coined by American pathologist Montrose Thomas Burrows.[15]

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.[1]

Modern usage

Cultured cells growing in growth medium

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.[16] 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.[17]

Plant tissue culture in particular is concerned with the growing of entire plants from small pieces of plant tissue, cultured in medium.[18]

Concepts in mammalian cell culture

Isolation of cells

Main article: 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.[19][20] 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 and stop dividing after a certain number of population doublings while generally retaining their viability (described as the Hayflick limit).

A bottle of DMEM cell culture medium

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).[21] 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,[22] and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.[23]

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.[24]

Cells can be grown either in suspension or adherent cultures.[25] 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).[26]

Cell culture basal media

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

Components of cell culture media

Component Function
Carbon source (glucose/glutamine) Source of energy
Amino acid Building blocks of protein
Vitamins Promote cell survival and growth
Balanced salt solution An isotonic mixture of ions to maintain optimum osmotic pressure within the cells and provide essential metal ions to act as cofactors for enzymatic reactions, cell adhesion etc.
Phenol red dye pH indicator. The color of phenol red changes from orange/red at pH 7–7.4 to yellow at acidic (lower) pH and purple at basic (higher) pH.
Bicarbonate /HEPES buffer It is used to maintain a balanced pH in the media

Typical Growth conditions

Parameter
Temperature 37  °C
CO2 5%
Relative Humidity 95%

Cell line cross-contamination

Main article: List of contaminated cell lines

Cell line cross-contamination can be a problem for scientists working with cultured cells.[27] Studies suggest anywhere from 15 to 20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.[28][29][30] 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.[31][32] 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.[31][33] 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.[34] ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.[35]

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.[35]

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.[36]

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.
  • 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.[37]

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.[38] A systematic bias in generated datasets was recently shown for CRISPR and RNAi gene silencing screens,[39] and for metabolic profiling of cancer cell lines.[38] 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[40] and Human Plasma Like Medium (HPLM),[41] 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 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.

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

Main article: Passaging

Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of trypsin-EDTA; however, other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture. Some cell cultures, such as RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers.

Transfection and transduction

Main articles: Transfection and 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.

Established human cell lines

Cultured HeLa 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.[42]

Further information: 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.

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[43] and, after this, they lose their ability to proliferate (a genetically determined event known as senescence).[44]

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 cells is used to expand the number of cells and differentiate the cells into various somatic cell types for transplantation.[45] Stem cell culture is also used to harvest the molecules and exosomes that the stem cells release for the purposes of therapeutic development.[46]

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.[47] 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.[48]

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. 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,[49] a concept that has led to discoveries in fields such as:

Cell culture in three dimensions

Cell culture in three dimensions has been touted as "Biology's New Dimension".[64] At present, the practice of cell culture remains based on varying combinations of single or multiple cell structures in 2D.[65] 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.[66][67][68] 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.[69] There are a variety of platforms used to facilitate the growth of three-dimensional cellular structures including scaffold systems such as hydrogel matrices[70] and solid scaffolds, and scaffold-free systems such as low-adhesion plates, nanoparticle facilitated magnetic levitation,[71] hanging drop plates,[72][73] and rotary cell culture. Culturing cells in 3D leads to wide variation in gene expression signatures and partly mimics tissues in the physiological states.[74] 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.[75] 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.[76]

3D cell culture in scaffolds

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.[17]

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.[77] 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 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, 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,[78][79] and novel adjuvants.[80]

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.[15][81]

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.[82]

Schematic representation of 2D culture, 3D culture, organ-on-a-chip and in vivo study

Cell culture in microfluidic device

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.[83][84]

Organ-on-a-chip

Main article: 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.[85][86] 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.[87]

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.[88][48][89]

Plant cell culture methods

Main article: 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.[citation needed]

Insect cell culture

Main article: 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.[90]

Bacterial and yeast culture methods

Main article: 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.[citation needed]

Viral culture methods

Main article: 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.[citation needed]

Common cell lines

Human cell lines
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
Rat tumor cell lines
Plant cell lines
Other species cell lines

List of cell lines

Cell line Meaning Organism Origin tissue Morphology Links
3T3-L1 "3-day transfer, inoculum 3 x 10^5 cells" Mouse Embryo Fibroblast ECACC Cellosaurus
4T1 Mouse Mammary gland ATCC Cellosaurus
1321N1 Human Brain Astrocytoma ECACC Cellosaurus
9L Rat Brain Glioblastoma ECACC Cellosaurus
A172 Human Brain Glioblastoma ECACC Cellosaurus
A20 Mouse B lymphoma B lymphocyte Cellosaurus
A253 Human Submandibular duct Head and neck carcinoma ATCC Cellosaurus
A2780 Human Ovary Ovarian carcinoma ECACC Cellosaurus
A2780ADR Human Ovary Adriamycin-resistant derivative of A2780 ECACC Cellosaurus
A2780cis Human Ovary Cisplatin-resistant derivative of A2780 ECACC Cellosaurus
A431 Human Skin epithelium Squamous cell carcinoma ECACC Cellosaurus
A549 Human Lung Lung carcinoma ECACC Cellosaurus
AB9 Zebrafish Fin Fibroblast ATCC Cellosaurus
AHL-1 Armenian Hamster Lung-1 Hamster Lung ECACC Archived 24 November 2021 at the Wayback Machine Cellosaurus
ALC Mouse Bone marrow Stroma PMID 2435412[91] Cellosaurus
B16 Mouse Melanoma ECACC Archived 24 November 2021 at the Wayback Machine Cellosaurus
B35 Rat Neuroblastoma ATCC Cellosaurus
BCP-1 Human PBMC HIV+ primary effusion lymphoma ATCC Cellosaurus
BEAS-2B Bronchial epithelium + Adenovirus 12-SV40 virus hybrid (Ad12SV40) Human Lung Epithelial ECACC Cellosaurus
bEnd.3 Brain Endothelial 3 Mouse Brain/cerebral cortex Endothelium Cellosaurus
BHK-21 Baby Hamster Kidney-21 Hamster Kidney Fibroblast ECACC Archived 24 November 2021 at the Wayback Machine Cellosaurus
BOSC23 Packaging cell line derived from HEK 293 Human Kidney (embryonic) Epithelium Cellosaurus
BT-20 Breast Tumor-20 Human Breast epithelium Breast carcinoma ATCC Cellosaurus
BxPC-3 Biopsy xenograft of Pancreatic Carcinoma line 3 Human Pancreatic adenocarcinoma Epithelial ECACC Cellosaurus
C2C12 Mouse Myoblast ECACC Cellosaurus
C3H-10T1/2 Mouse Embryonic mesenchymal cell line ECACC Cellosaurus
C6 Rat Brain astrocyte Glioma ECACC Cellosaurus
C6/36 Insect - Asian tiger mosquito Larval tissue ECACC Cellosaurus
Caco-2 Human Colon Colorectal carcinoma ECACC Cellosaurus
Cal-27 Human Tongue Squamous cell carcinoma ATCC Cellosaurus
Calu-3 Human Lung Adenocarcinoma ATCC Cellosaurus
CGR8 Mouse Embryonic stem cells ECACC Cellosaurus
CHO Chinese Hamster Ovary Hamster Ovary Epithelium ECACC Archived 29 October 2021 at the Wayback Machine Cellosaurus
CML T1 Chronic myeloid leukemia T lymphocyte 1 Human CML acute phase T cell leukemia DSMZ Cellosaurus
CMT12 Canine Mammary Tumor 12 Dog Mammary gland Epithelium Cellosaurus
COR-L23 Human Lung Lung carcinoma ECACC Cellosaurus
COR-L23/5010 Human Lung Lung carcinoma ECACC Cellosaurus
COR-L23/CPR Human Lung Lung carcinoma ECACC Cellosaurus
COR-L23/R23- Human Lung Lung carcinoma ECACC Cellosaurus
COS-7 Cercopithecus aethiops, origin-defective SV-40 Old World monkey - Cercopithecus aethiops (Chlorocebus) Kidney Fibroblast ECACC Cellosaurus
COV-434 Human Ovary Ovarian granulosa cell carcinoma PMID 8436435[92] ECACC Cellosaurus
CT26 Mouse Colon Colorectal carcinoma Cellosaurus
D17 Dog Lung metastasis Osteosarcoma ATCC Cellosaurus
DAOY Human Brain Medulloblastoma ATCC Cellosaurus
DH82 Dog Histiocytosis Monocyte/macrophage ECACC Cellosaurus
DU145 Human Androgen insensitive prostate carcinoma ATCC Cellosaurus
DuCaP Dura mater cancer of the Prostate Human Metastatic prostate carcinoma Epithelial PMID 11317521[93] Cellosaurus
E14Tg2a Mouse Embryonic stem cells ECACC Cellosaurus
EL4 Mouse T cell leukemia ECACC Cellosaurus
EM-2 Human CML blast crisis Ph+ CML line DSMZ Cellosaurus
EM-3 Human CML blast crisis Ph+ CML line DSMZ Cellosaurus
EMT6/AR1 Mouse Mammary gland Epithelial-like ECACC Cellosaurus
EMT6/AR10.0 Mouse Mammary gland Epithelial-like ECACC Cellosaurus
FM3 Human Lymph node metastasis Melanoma ECACC Cellosaurus
GL261 Glioma 261 Mouse Brain Glioma Cellosaurus
H1299 Human Lung Lung carcinoma ATCC Cellosaurus
HaCaT Human Skin Keratinocyte CLS Cellosaurus
HCA2 Human Colon Adenocarcinoma ECACC Cellosaurus
HEK 293 Human Embryonic Kidney 293 Human Kidney (embryonic) Epithelium ECACC Cellosaurus
HEK 293T HEK 293 derivative Human Kidney (embryonic) Epithelium ECACC Cellosaurus
HeLa "Henrietta Lacks" Human Cervix epithelium Cervical carcinoma ECACC Cellosaurus
Hepa1c1c7 Clone 7 of clone 1 hepatoma line 1 Mouse Hepatoma Epithelial ECACC Cellosaurus
Hep G2 Human Liver Hepatoblastoma ECACC Cellosaurus
High Five Insect (moth) - Trichoplusia ni Ovary Cellosaurus
HL-60 Human Leukemia-60 Human Blood Myeloblast ECACC Cellosaurus
HT-1080 Human Fibrosarcoma ECACC Cellosaurus
HT-29 Human Colon epithelium Adenocarcinoma ECACC Cellosaurus
J558L Mouse Myeloma B lymphocyte cell ECACC Cellosaurus
Jurkat Human White blood cells T cell leukemia ECACC Cellosaurus
JY Human Lymphoblastoid EBV-transformed B cell ECACC Cellosaurus
K562 Human Lymphoblastoid CML blast crisis ECACC Cellosaurus
KBM-7 Human Lymphoblastoid CML blast crisis Cellosaurus
KCL-22 Human Lymphoblastoid CML DSMZ Cellosaurus
KG1 Human Lymphoblastoid AML ECACC Cellosaurus
Ku812 Human Lymphoblastoid Erythroleukemia ECACC Cellosaurus
KYO-1 Kyoto-1 Human Lymphoblastoid CML DSMZ Cellosaurus
L1210 Mouse Lymphocytic leukemia Ascitic fluid ECACC Cellosaurus
L243 Mouse Hybridoma Secretes L243 mAb (against HLA-DR) ATCC Cellosaurus
LNCaP Lymph Node Cancer of the Prostate Human Prostatic adenocarcinoma Epithelial ECACC Cellosaurus
MA-104 Microbiological Associates-104 African Green Monkey Kidney Epithelial Cellosaurus
MA2.1 Mouse Hybridoma Secretes MA2.1 mAb (against HLA-A2 and HLA-B17) ATCC Cellosaurus
Ma-Mel 1, 2, 3....48 Human Skin A range of melanoma cell lines ECACC Archived 24 November 2021 at the Wayback Machine Cellosaurus
MC-38 Mouse Colon-38 Mouse Colon Adenocarcinoma Cellosaurus
MCF-7 Michigan Cancer Foundation-7 Human Breast Invasive breast ductal carcinoma ER+, PR+ ECACC Cellosaurus
MCF-10A Michigan Cancer Foundation-10A Human Breast epithelium ATCC Cellosaurus
MDA-MB-157 M.D. Anderson - Metastatic Breast-157 Human Pleural effusion metastasis Breast carcinoma ECACC Cellosaurus
MDA-MB-231 M.D. Anderson - Metastatic Breast-231 Human Pleural effusion metastasis Breast carcinoma ECACC Cellosaurus
MDA-MB-361 M.D. Anderson - Metastatic Breast-361 Human Melanoma (contaminated by M14) ECACC Cellosaurus
MDA-MB-468 M.D. Anderson - Metastatic Breast-468 Human Pleural effusion metastasis Breast carcinoma ATCC Cellosaurus
MDCK II Madin Darby Canine Kidney II Dog Kidney Epithelium ECACC Cellosaurus
MG63 Human Bone Osteosarcoma ECACC Cellosaurus
MIA PaCa-2 Human Prostate Pancreatic Carcinoma ATCC Cellosaurus
MOR/0.2R Human Lung Lung carcinoma ECACC Cellosaurus
Mono-Mac-6 Human White blood cells Myeloid metaplasic AML DSMZ Cellosaurus
MRC-5 Medical Research Council cell strain 5 Human Lung (fetal) Fibroblast ECACC Archived 24 November 2021 at the Wayback Machine Cellosaurus
MTD-1A Mouse Epithelium Cellosaurus
MyEnd Myocardial Endothelial Mouse Endothelium Cellosaurus
NCI-H69 Human Lung Lung carcinoma ECACC Cellosaurus
NCI-H69/CPR Human Lung Lung carcinoma ECACC Cellosaurus
NCI-H69/LX10 Human Lung Lung carcinoma ECACC Cellosaurus
NCI-H69/LX20 Human Lung Lung carcinoma ECACC Cellosaurus
NCI-H69/LX4 Human Lung Lung carcinoma ECACC Cellosaurus
Neuro-2a Mouse Nerve/neuroblastoma Neuronal stem cells ECACC Cellosaurus
NIH-3T3 NIH, 3-day transfer, inoculum 3 x 105 cells Mouse Embryo Fibroblast ECACC Cellosaurus
NALM-1 Human Peripheral blood Blast-crisis CML ATCC Cellosaurus
NK-92 Human Leukemia/lymphoma ATCC Cellosaurus
NTERA-2 Human Lung metastasis Embryonal carcinoma ECACC Cellosaurus
NW-145 Human Skin Melanoma ESTDAB Archived 2011-11-16 at the Wayback Machine Cellosaurus
OK Opossum Kidney Virginia opossum - Didelphis virginiana Kidney ECACC Cellosaurus
OPCN / OPCT cell lines Human Prostate Range of prostate tumour lines Cellosaurus
P3X63Ag8 Mouse Myeloma ECACC Cellosaurus
PANC-1 Human Duct Epithelioid Carcinoma ATCC Cellosaurus
PC12 Rat Adrenal medulla Pheochromocytoma ECACC Cellosaurus
PC-3 Prostate Cancer-3 Human Bone metastasis Prostate carcinoma ECACC Cellosaurus
Peer Human T cell leukemia DSMZ Cellosaurus
PNT1A Human Prostate SV40-transformed tumour line ECACC Cellosaurus
PNT2 Human Prostate SV40-transformed tumour line ECACC Cellosaurus
Pt K2 The second cell line derived from Potorous tridactylis Long-nosed potoroo - Potorous tridactylus Kidney Epithelial ECACC Cellosaurus
Raji Human B lymphoma Lymphoblast-like ECACC Cellosaurus
RBL-1 Rat Basophilic Leukemia-1 Rat Leukemia Basophil cell ECACC Cellosaurus
RenCa Renal Carcinoma Mouse Kidney Renal carcinoma ATCC Cellosaurus
RIN-5F Mouse Pancreas ECACC Cellosaurus
RMA-S Mouse T cell tumour Cellosaurus
S2 Schneider 2 Insect - Drosophila melanogaster Late stage (20–24 hours old) embryos ATCC Cellosaurus
SaOS-2 Sarcoma OSteogenic-2 Human Bone Osteosarcoma ECACC Cellosaurus
Sf21 Spodoptera frugiperda 21 Insect (moth) - Spodoptera frugiperda Ovary ECACC Cellosaurus
Sf9 Spodoptera frugiperda 9 Insect (moth) - Spodoptera frugiperda Ovary ECACC Cellosaurus
SH-SY5Y Human Bone marrow metastasis Neuroblastoma ECACC Cellosaurus
SiHa Human Cervix epithelium Cervical carcinoma ATCC Cellosaurus
SK-BR-3 Sloan-Kettering Breast cancer 3 Human Breast Breast carcinoma DSMZ Cellosaurus
SK-OV-3 Sloan-Kettering Ovarian cancer 3 Human Ovary Ovarian carcinoma ECACC Cellosaurus
SK-N-SH Human Brain Epithelial ATCC Cellosaurus
T2 Human T cell leukemia/B cell line hybridoma ATCC Cellosaurus
T-47D Human Breast Breast ductal carcinoma ECACC Cellosaurus
T84 Human Lung metastasis Colorectal carcinoma ECACC Cellosaurus
T98G Human Glioblastoma-astrocytoma Epithelium ECACC Cellosaurus
THP-1 Human Monocyte Acute monocytic leukemia ECACC Cellosaurus
U2OS Human Osteosarcoma Epithelial ECACC Cellosaurus
U373 Human Glioblastoma-astrocytoma Epithelium ECACC Archived 24 November 2021 at the Wayback Machine Cellosaurus
U87 Human Glioblastoma-astrocytoma Epithelial-like ECACC Cellosaurus
U937 Human Leukemic monocytic lymphoma ECACC Cellosaurus
VCaP Vertebral Cancer of the Prostate Human Vertebra metastasis Prostate carcinoma ECACC Cellosaurus
Vero From Esperanto: verda (green, for green monkey) reno (kidney) African green monkey - Chlorocebus sabaeus Kidney epithelium ECACC Cellosaurus
VG-1 Human Primary effusion lymphoma Cellosaurus
WM39 Human Skin Melanoma ESTDAB Cellosaurus
WT-49 Human Lymphoblastoid ECACC Cellosaurus
YAC-1 Mouse Lymphoma ECACC Cellosaurus
YAR Human Lymphoblastoid EBV-transformed B cell Human Immunology[94] ECACC Cellosaurus

See also

References and notes

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Further reading

Library resources about
Cell culture

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