Chinese hamster ovary cell

Last updated
CHO cells adhered to a surface, seen under phase-contrast microscopy Cho cells adherend2.jpg
CHO cells adhered to a surface, seen under phase-contrast microscopy

Chinese hamster ovary (CHO) cells are a family of immortalized cell lines [1] derived from epithelial cells of the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. [1] [2] They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, and particularly since the 1980s to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics. [2]

Contents

History

Chinese hamsters had been used in research since 1919, where they were used in place of mice for typing pneumococci. They were subsequently found to be excellent vectors for transmission of kala-azar (visceral leishmaniasis), facilitating Leishmania research. [3] [4]

In 1948, the Chinese hamster was first used in the United States for breeding in research laboratories. In 1957, Theodore T. Puck 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. [3] [5]

The thrombolytic medication against myocardial infarction alteplase (Activase) was approved by the US Food and Drug Administration in 1987. It was the first commercially available recombinant protein produced from CHO cells. [3] [6] CHO cells continue to be the most widely used manufacturing approach for recombinant protein therapeutics and prophylactic agents. [7] [8] In 2019, six of the 10 best selling drugs were made in CHO cells. [9]

Properties

All CHO cell lines are deficient in proline synthesis. [10] Also, CHO cells do not express the epidermal growth factor receptor (EGFR), which makes them ideal in the investigation of various EGFR mutations. [11]

Furthermore, Chinese hamster ovary cells are able to produce proteins 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. [12]

Having a very low chromosome number (2n=22) for a mammal, the Chinese hamster is also a good model for radiation cytogenetics and tissue culture. [13]

Variants

Since the original CHO cell line was described in 1956, many variants of the cell line have been developed for various purposes. [10] [ additional citation(s) needed ] In 1957, CHO-K1 was generated from a single clone of CHO cells. [14] According to an industry source, however, scientist Theodore Puck first isolated CHO-K1 in 1968. [1] Puck and colleagues reported starting a cell line of Chinese hamster ovarian origin in 1957. [15] [16] Variants of K1 include the deposits in ATCC, ECACC, and a version adapted for growth in protein-free medium. [14]

CHO-K1 was mutagenized in the 1970s with ethyl methanesulfonate to generate a cell line lacking dihydrofolate reductase (DHFR) activity, referred to as CHO-DXB11 (also referred to as CHO-DUKX). [17] However, these cells, when mutagenized, could revert to DHFR activity, making their utility for research somewhat limited. [17] Subsequently in 1983, CHO cells were mutagenized with gamma radiation to yield a cell line in which both alleles of the DHFR locus were completely eliminated, termed CHO-DG44. [18] These DHFR-deficient strains require glycine, hypoxanthine, and thymidine for growth. [18] 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. This sort of system requires no special mutation, so that non-DHFR-deficient host cell culture 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. For example, the three K1 offspring cultures available in 2013 each have significant accumulated mutations compared to each other. [14] 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. [10] [14] The complex genetics of CHO cells and the issues concerning clonal derivation of cell population was extensively discussed. [19] [20]

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 of the gene of interest and the DHFR gene into a single mammalian expression system. The plasmid DNA carrying the two genes is then transfected into cells, and the cells are grown under selective conditions in a thymidine-lacking medium. Surviving cells will have the exogenous DHFR gene along with the gene of interest integrated in its genome. [21] [22] The growth rate and the level of recombinant protein 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. [23]

Industrial use

CHO cells are the most common mammalian cell line used for mass production of therapeutic proteins such as monoclonal antibodies, used in 70% of therapeutic mAbs. [2] They can produce recombinant protein on the scale of 3–10 grams per liter of culture. [10] Products of CHO cells are suitable for human applications, as these mammalian cells perform human-like post-translational modifications to recombinant proteins, which is key to the functioning of several proteins. [24]

See also

Related Research Articles

<span class="mw-page-title-main">Dihydrofolate reductase</span> Mammalian protein found in Homo sapiens

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. In humans, the DHFR enzyme is encoded by the DHFR gene. It is found in the q14.1 region of chromosome 5.

<span class="mw-page-title-main">Protein production</span>

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, are an immortalised cell line derived from HEK cells isolated from a female fetus in the 1970s.

<i>Komagataella</i> Genus of fungus used industrially and as model organism

Komagataella is a methylotrophic yeast within the order Saccharomycetales. It was found in the 1960s as Pichia pastoris, with its feature of using methanol as a source of carbon and energy. In 1995, P. pastoris was reassigned into the sole representative of genus Komagataella, becoming Komagataella phaffii. Later studies have further distinguished new species in this genus, resulting in a total of 7 recognized species. It is not uncommon to see the old name still in use in the context of protein production, as of 2023; in less formal use, the yeast may confusingly be referred to as pichia.

Theodore Thomas Puck 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 PhD work was on the laws governing the impact of an electron upon an atom and his doctoral adviser was James Franck. During WW II Puck stayed at the University of Chicago. There he worked in the laboratory of Oswald H. Robertson on the study of how bacteria and viruses can spread through the air and on dust particles. After a postdoc position in the laboratory of Renato Dulbecco, Puck was recruited in 1948 to establish and chair the University of Colorado Medical School's department of biophysics. He retired from the University of Colorado Medical School in 1995 as professor emeritus, but continued to do laboratory work there until a few weeks before his death.

<span class="mw-page-title-main">Vero cell</span> Cell lineage used in cell cultures

Vero cells are a lineage of cells used in cell cultures. The 'Vero' lineage was isolated from kidney epithelial cells extracted from an African green monkey. The lineage was developed on 27 March 1962 by Yasumura and Kawakita at the Chiba University in Chiba, Japan. The original cell line was named Vero after an abbreviation of verdareno, which means 'green kidney' in Esperanto, while vero itself means 'truth' in Esperanto.

<span class="mw-page-title-main">COS cells</span> Cell lines derived from monkey kidney tissue

COS are fibroblast-like cell lines derived from monkey kidney tissue. COS cells are obtained by immortalizing CV-1 cells with a version of the SV40 virus that can produce large T antigen but has a defect in genomic replication. The CV-1 cell line in turn was derived from the kidney of the African green monkey.

<span class="mw-page-title-main">Biotechnology in pharmaceutical manufacturing</span>

Biotechnology is the use of living organisms to develop useful products. Biotechnology is often used in pharmaceutical manufacturing. Notable examples include the use of bacteria to produce things such as insulin or human growth hormone. Other examples include the use of transgenic pigs for the creation of hemoglobin in use of humans.

<span class="mw-page-title-main">CLIC1</span> Protein-coding gene in the species Homo sapiens

Chloride intracellular channel protein 1 is a protein that in humans is encoded by the CLIC1 gene.

<span class="mw-page-title-main">40S ribosomal protein S14</span> Protein-coding gene in the species Homo sapiens

40S ribosomal protein S14 is a protein that in humans is encoded by the RPS14 gene.

<span class="mw-page-title-main">ADK (gene)</span> Protein-coding gene in the species Homo sapiens

Adenosine kinase is an enzyme that in humans is encoded by the ADK gene.

<span class="mw-page-title-main">REPIN1</span> Protein-coding gene in the species Homo sapiens

Replication initiator 1 is a protein that in humans is encoded by the REPIN1 gene. The protein helps enable RNA binding activity as a replication initiation-region protein. The make up of REPIN 1 include three zinc finger hand clusters that organize polydactyl zinc finger proteins containing 15 zinc finger DNA- binding motifs. It has also been predicted to help in regulation of transcription via RNA polymerase II with it being located in the nucleoplasm. Expression of this protein has been seen in the colon, spleen, kidney, and 23 other tissues within the human body throughout.

A subunit vaccine is a vaccine that contains purified parts of the pathogen that are antigenic, or necessary to elicit a protective immune response. Subunit vaccine can be made from dissembled viral particles in cell culture or recombinant DNA expression, in which case it is a recombinant subunit vaccine.

High Five (BTI-Tn-5B1-4) is an insect cell line that originated from the ovarian cells of the cabbage looper, Trichoplusia ni. It was developed by the Boyce Thompson Institute for Plant Research.

Schneider 2 cells, usually abbreviated as S2 cells, are one of the most commonly used Drosophila melanogaster cell lines. S2 cells were derived from a primary culture of late stage Drosophila melanogaster embryos by Dr. Imogene Schneider, likely from a macrophage-like lineage.

NS0 cells are a model cell line derived from the nonsecreting murine myeloma used in biomedical research and commercially in the production of therapeutic proteins. The cell line is a cholesterol-dependent cell line that was generated from a subline of NSI/1 which produced only the light chain but no heavy chain.

Transient expression, more frequently referred to "transient gene expression", is the temporary expression of genes that are expressed for a short time after nucleic acid, most frequently plasmid DNA encoding an expression cassette, has been introduced into eukaryotic cells with a chemical delivery agent like calcium phosphate (CaPi) or polyethyleneimine (PEI). However, unlike "stable expression," the foreign DNA does not fuse with the host cell DNA, resulting in the inevitable loss of the vector after several cell replication cycles. The majority of transient gene expressions are done with cultivated animal cells. The technique is also used in plant cells; however, the transfer of nucleic acids into these cells requires different methods than those with animal cells. In both plants and animals, transient expression should result in a time-limited use of transferred nucleic acids, since any long-term expression would be called "stable expression."

<span class="mw-page-title-main">June Biedler</span> American scientist

June Biedler was an American scientist primarily known for her discovery of proteins that lead to resistance of cancer cells to chemotherapy. Her work has been crucial for an understanding of both the development of drug resistance and also for strategies to circumvent such resistance. In addition, Biedler made important contributions to an understanding of the molecular mechanisms of neuroblastoma development, particularly of the role of the N-myc oncogene in the genesis of neuroblastoma

<span class="mw-page-title-main">Cell engineering</span>

Cell engineering is the purposeful process of adding, deleting, or modifying genetic sequences in living cells to achieve biological engineering goals such as altering cell production, changing cell growth and proliferation requirements, adding or removing cell functions, and many more. Cell engineering often makes use of DNA technology to achieve these modifications as well as closely related tissue engineering methods. Cell engineering can be characterized as an intermediary level in the increasingly specific disciplines of biological engineering which includes organ engineering, tissue engineering, protein engineering, and genetic engineering.

References

  1. 1 2 3 Eberle, Christian (3 May 2022). "CHO cells – 7 facts about the cell line derived from the ovary of the Chinese hamster". evitria. Retrieved 30 January 2024.
  2. 1 2 3 Wurm FM (2004). "Production of recombinant protein therapeutics in cultivated mammalian cells". Nature Biotechnology. 22 (11): 1393–1398. doi:10.1038/nbt1026. PMID   15529164. S2CID   20428452.
  3. 1 2 3 "Vital Tools A Brief History of CHO Cells" (PDF). LSF Magazine. Winter 2015. pp. 38–47. Retrieved 5 April 2023.
  4. Young C, Smyly H, Brown C (March 1924). "Experimental kala-azar in a hamster". Experimental Biology and Medicine. 21 (6): 357–359. doi:10.3181/00379727-21-182. ISSN   1535-3702.
  5. Fanelli, Alex (2016). "CHO Cells" . Retrieved 28 November 2017.
  6. Du C; Webb C (2011). "Cellular Systems". Comprehensive Biotechnology. Elsevier. pp. 11–23. doi:10.1016/b978-0-08-088504-9.00080-5. ISBN   9780080885049.
  7. Tihanyi B, Nyitray L (December 2020). "Recent advances in CHO cell line development for recombinant protein production". Drug Discovery Today . 38: 25–34. doi:10.1016/j.ddtec.2021.02.003. hdl: 10831/82853 . PMID   34895638. However, 70% of biologics, and almost all mAbs, are produced in Chinese hamster ovary (CHO) cells, as the most commonly used and preferred hosts for biopharmaceutical protein production.
  8. Liang K, Luo H, Li Q (2023). "Enhancing and stabilizing monoclonal antibody production by Chinese hamster ovary (CHO) cells with optimized perfusion culture strategies". Frontiers in Bioengineering and Biotechnology . 11: 1112349. doi: 10.3389/fbioe.2023.1112349 . PMC   9895834 . PMID   36741761. Since 2016, about 70% of all rBPs and mAbs were produced from Chinese hamster ovary (CHO) cell lines
  9. Li ZM, Fan ZL, Wang XY, Wang TY (2022). "Factors Affecting the Expression of Recombinant Protein and Improvement Strategies in Chinese Hamster Ovary Cells". Frontiers in Bioengineering and Biotechnology. 10: 880155. doi: 10.3389/fbioe.2022.880155 . PMC   9289362 . PMID   35860329. By 2019, all six of the top ten best-selling drugs were produced in CHO cells (Urquhart, 2020).
  10. 1 2 3 4 Wurm FM; Hacker D (2011). "First CHO genome". Nature Biotechnology. 29 (8): 718–20. doi:10.1038/nbt.1943. PMID   21822249. S2CID   8422581.
  11. Ahsan, A.; S. M. Hiniker; M. A. Davis; T. S. Lawrence; M. K. Nyati (2009). "Role of Cell Cycle in Epidermal Growth Factor Receptor Inhibitor-Mediated Radiosensitization". Cancer Research. 69 (12): 5108–5114. doi:10.1158/0008-5472.CAN-09-0466. PMC   2697971 . PMID   19509222.
  12. "CHO cells - 7 facts about the cell line derived from the ovary of the Chinese hamster". evitria AG. 3 May 2022.
  13. Tjio J. H.; Puck T. T. (1958). "Genetics of somatic mammalian cells. II. chromosomal constitution of cells in tissue culture". J. Exp. Med. 108 (2): 259–271. doi:10.1084/jem.108.2.259. PMC   2136870 . PMID   13563760.
  14. 1 2 3 4 Lewis NE; Liu X; Li Y; Nagarajan H; Yerganian G; O'Brien E; et al. (2013). "Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome". Nature Biotechnology. 31 (8): 759–765. doi: 10.1038/nbt.2624 . PMID   23873082.
  15. Puck TT, Cieciura SJ, Robinson A (1958). "Genetics of Somatic Mammalian Cells: III. Long-Term Cultivation of Euploid Cells from Human and Animal Subjects". Journal of Experimental Biology. 108 (6): 945–956. doi: 10.1084/jem.108.6.945 . PMC   2136918 . PMID   13598821.
  16. Ham RG (1965). "Clonal Growth of Mammalian Cells in a Chemically Defined, Synthetic Medium". Proceedings of the National Academy of Sciences. 53 (2): 288–293. Bibcode:1965PNAS...53..288H. doi: 10.1073/pnas.53.2.288 . PMC   219509 . PMID   14294058.
  17. 1 2 Urlaub G; Chasin LA (July 1980). "Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity". Proceedings of the National Academy of Sciences of the United States of America. 77 (7): 4216–4220. Bibcode:1980PNAS...77.4216U. doi: 10.1073/pnas.77.7.4216 . PMC   349802 . PMID   6933469.
  18. 1 2 Urlaub G; Kas E; Carothers AD; Chasin LA (June 1983). "Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells". Cell. 33 (2): 405–412. doi: 10.1016/0092-8674(83)90422-1 . PMID   6305508.
  19. Wurm, Florian; Wurm, Maria (2017). "Cloning of CHO cells, productivity and genetic stability – a discussion". Processes. 5 (4): 20. doi: 10.3390/pr5020020 .
  20. Reinhart, D; Damjanovic, L; Kaisermayer, C; Sommeregger, W; Gili, A; Gasselhuber, B; Castan, A; Mayrhofer, P; Grünwald-Gruber, C; Kunert, R (March 2019). "Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis". Biotechnology Journal. 14 (3): e1700686. doi: 10.1002/biot.201700686 . PMID   29701329. S2CID   13844297.
  21. Lee F; Mulligan R; Berg P; Ringold G (19 November 1981). "Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids". Nature. 294 (5838): 228–232. Bibcode:1981Natur.294..228L. doi:10.1038/294228a0. PMID   6272123. S2CID   2501119.
  22. Kaufman RJ; Sharp PA (25 August 1982). "Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene". Journal of Molecular Biology. 159 (4): 601–621. doi:10.1016/0022-2836(82)90103-6. PMID   6292436.
  23. "General Cell Collection: CHO-K1". Hpacultures.org.uk. 2000-01-01. Retrieved 2013-05-21.
  24. Tingfeng, Lai; et al. (2013). "Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production". Pharmaceuticals. 6 (5): 579–603. doi: 10.3390/ph6050579 . PMC   3817724 . PMID   24276168.