Chinese hamster ovary (CHO) cells are an

epithelial Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellu ...

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 cell ...

derived from the

ovary The ovary is an organ in the female reproductive system that produces an ovum. When released, this travels down the fallopian tube into the uterus, where it may become fertilized by a sperm. There is an ovary () found on each side of the body. ...

of the

Chinese hamster The Chinese hamster (''Cricetulus griseus'' or ''Cricetulus barabensis griseus'') is a rodent in the genus ''Cricetulus ''Cricetulus'' is a genus of rodent in the family Cricetidae (voles and hamsters); it has seven member species that inhab ...

, often used in

biological Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary in ...

and

medical research Medical research (or biomedical research), also known as experimental medicine, encompasses a wide array of research, extending from " basic research" (also called ''bench science'' or ''bench research''), – involving fundamental scienti ...

and commercially in the production of recombinant therapeutic

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

History

Chinese hamsters had been used in research since 1919, where they were used in place of mice for typing

pneumococci ''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, alpha-hemolytic (under aerobic conditions) or beta-hemolytic (under anaerobic conditions), aerotolerant anaerobic member of the genus Streptococcus. They are ...

. They were subsequently found to be excellent vectors for transmission of kala-azar (

visceral leishmaniasis Visceral leishmaniasis (VL), also known as kala-azar ( Hindi: kālā āzār, "black sickness") or "black fever", is the most severe form of leishmaniasis and, without proper diagnosis and treatment, is associated with high fatality. Leishmanias ...

), facilitating ''

Leishmania ''Leishmania'' is a parasitic protozoan, a single-celled organism of the genus '' Leishmania'' that are responsible for the disease leishmaniasis. They are spread by sandflies of the genus ''Phlebotomus'' in the Old World, and of the genus ' ...

'' research. In 1948, the Chinese hamster was first used in the United States for breeding in research laboratories. In 1957,

Theodore T. Puck Theodore Thomas Puck (September 24, 1916 – November 6, 2005) was an American geneticist born in Chicago, Illinois. He attended Chicago public schools and obtained his bachelors, masters, and doctoral degree from the University of Chicago. His ...

obtained a female Chinese hamster from Dr. George Yerganian's laboratory at the Boston Cancer Research Foundation and used it to derive the original Chinese hamster ovary (CHO) cell line. Since then, CHO cells have been a cell line of choice because of their rapid growth in suspension culture and high protein production. Having a very low

chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins ar ...

number (2n=22) for a

mammal Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur ...

, the Chinese hamster is also a good model for radiation cytogenetics and tissue culture.

Properties

All CHO cell lines are deficient in

proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...

synthesis. Also, CHO cells do not express the

epidermal growth factor receptor The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. The epidermal growth factor re ...

(EGFR), which makes them ideal in the investigation of various EGFR mutations. Furthermore, Chinese hamster ovary cells are able to produce

with complex glycosylations, post-translational modifications (PTMs) similar to those produced in humans. They are easily growable in large-scale cultures and have great viability, which is why they are ideal for GMP protein production. Also, CHO cells are tolerant to variations in parameters, be it oxygen levels, pH-value, temperature or cell density.

Variants

Since the original CHO cell line was described in 1956, many variants of the cell line have been developed for various purposes. In 1957, CHO-K1 was generated from a single clone of CHO cells, CHO-K1 was mutagenized with ethyl methanesulfonate to generate a cell line lacking

dihydrofolate reductase Dihydrofolate reductase, or DHFR, is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as an electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1-carbon transfer chemistry ...

(DHFR) activity, referred to as CHO-DXB11 (also referred to as CHO-DUKX). However, these cells, when mutagenized, could revert to DHFR activity, making their utility for research somewhat limited. Subsequently, CHO cells were mutagenized with

gamma radiation A gamma ray, also known as gamma radiation (symbol γ or \gamma), is a penetrating form of electromagnetic radiation arising from the radioactive decay of atomic nuclei. It consists of the shortest wavelength electromagnetic waves, typically s ...

to yield a cell line in which both

alleles An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chrom ...

of the DHFR locus were completely eliminated, termed CHO-DG44. These DHFR-deficient strains require

glycine Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid ( carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinog ...

hypoxanthine Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids, where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-hydroxypurine. Hyp ...

, and thymidine for growth. Cell lines with mutated DHFR are useful for genetic manipulation as cells transfected with a gene of interest along with a functional copy of the ''DHFR'' gene can easily be screened for in thymidine-lacking media. Due to this, CHO cells lacking DHFR are the most widely used CHO cells for industrial protein production. More recently, other selection systems have become popular and with vector systems that can more efficiently target active chromatin in CHO cells, antibiotic selection (puromycin) can be used as well to generate recombinant cells expressing proteins at high level. For this, other host cells, still using the names as applied during the years from the 1960s to 1980s (CHO-K1, CHO-S, CHO-Pro minus, etc) have been found to produce excellent levels of proteins. Since CHO cells have a very high propensity of genetic instability (like all immortalised cells) one should not assume that the names applied indicate their usefulness for manufacturing purposes. Most, if not all industrially used CHO cell lines are now cultivated in animal component free media or in chemically defined media, and are used in large scale bioreactors under suspension culture. The complex genetics of CHO cells and the issues concerning clonal derivation of cell population was extensively discussed.

Genetic manipulation

Much of the genetic manipulation done in CHO cells is done in cells lacking DHFR enzyme. This genetic selection scheme remains one of the standard methods to establish transfected CHO cell lines for the production of recombinant therapeutic proteins. The process begins with the

molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word '' cloning'' refers to the fact that the meth ...

of the gene of interest and the ''DHFR'' gene into a single mammalian

expression system 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 ...

. The

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...

DNA carrying the two genes is then transfected into cells, and the cells are grown under selective conditions in a thymidine-lacking

medium Medium may refer to: Science and technology Aviation * Medium bomber, a class of war plane * Tecma Medium, a French hang glider design Communication * Media (communication), tools used to store and deliver information or data * Medium ...

. Surviving cells will have the

exogenous In a variety of contexts, exogeny or exogeneity () is the fact of an action or object originating externally. It contrasts with endogeneity or endogeny, the fact of being influenced within a system. Economics In an economic model, an exogen ...

''DHFR'' gene along with the gene of interest integrated in its

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...

. The growth rate and the level of

recombinant protein Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fo ...

production of each cell line varies widely. To obtain a few stably transfected cell lines with the desired phenotypic characteristics, evaluating several hundred candidate cell lines may be necessary. The CHO and CHO-K1 cell lines can be obtained from a number of biological resource centres such as the European Collection of Cell Cultures, which is part of the Health Protection Agency Culture Collections. These organizations also maintain data, such as growth curves, timelapse videos of growth, images, and subculture routine information.

Industrial use

CHO cells are the most common mammalian cell line used for mass production of therapeutic proteins. They can produce recombinant protein on the scale of 3-10 grams per liter of culture. Products of CHO cells are suitable for human applications, as they allow post-translational modifications to recombinant proteins which can function in humans.

References

External links

Chinese Hamster Genome Database

Recombinant Protein Therapeutics from CHO Cells — 20 Years and Counting
*{{cite journal , vauthors=Puck TT, Cieciura SJ, Robinson A , title=Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects , journal=J. Exp. Med. , volume=108 , issue=6 , pages=945–56 , date=December 1958 , pmid=13598821 , pmc=2136918 , doi=10.1084/jem.108.6.945
Cellosaurus entry for CHOCellosaurus entry for CHO-K1Cellosaurus entry for CHO-DG44Cellosaurus entry for CHO-DXB11
Rodent cell lines