HEK 293 cells

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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. [1] [2]

Contents

The HEK 293 cell line has been widely used in research for decades due to its reliable and fast growth and propensity for transfection. The cell line is used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy as well as safety testing for a vast array of chemicals.

History

HEK 293 cells were generated in 1973 by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex van der Eb's laboratory in Leiden, the Netherlands. The cells were obtained from a single, aborted or miscarried fetus, the precise origin of which is unclear. [3] [2] The cells were cultured by van der Eb; the transfection with adenoviral DNA was performed by Frank Graham, a post-doc in van der Eb's lab. They were published in 1977 after Graham left Leiden for McMaster University. [4] They are called HEK since they originated in human embryonic kidney cultures, while the number 293 came from Graham's habit of numbering his experiments; the original HEK 293 cell clone was from his 293rd experiment. Graham performed the transfection a total of eight times, obtaining just one clone of cells that were cultured for several months. After presumably adapting to tissue culture, cells from this clone developed into the relatively stable HEK 293 line.

Subsequent analysis has shown that the transformation was brought about by inserting ~4.5 kilobases from the left arm of the adenoviral genome, which became incorporated into human chromosome 19. [5]

For many years it was assumed that HEK 293 cells were generated by transformation of either a fibroblastic, endothelial or epithelial cell, all of which are abundant in kidneys. However, the original adenovirus transformation was inefficient, suggesting that the cell that finally produced the HEK 293 line may have been unusual in some fashion. Graham and coworkers provided evidence that HEK 293 cells and other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons, suggesting that the adenovirus preferentially transformed a neuronal lineage cell in the original kidney culture. [6]

A comprehensive study of the genomes and transcriptomes of HEK 293 and five derivative cell lines compared the HEK 293 transcriptome with that of human kidney, adrenal, pituitary and central nervous tissue. [7] The HEK 293 pattern most closely resembled that of adrenal cells, which have many neuronal properties. Given the location of the adrenal gland (adrenal means "next to the kidney"), a few adrenal cells could plausibly have appeared in an embryonic kidney derived culture, and could be preferentially transformed by adenovirus. Adenoviruses transform neuronal lineage cells much more efficiently than typical human kidney epithelial cells. [6] An embryonic adrenal precursor cell therefore seems the most likely origin cell of the HEK 293 line. As a consequence, HEK 293 cells should not be used as an in vitro model of typical kidney cells.

HEK 293 cells have a complex karyotype, exhibiting two or more copies of each chromosome and with a modal chromosome number of 64. They are described as hypotriploid, containing less than three times the number of chromosomes of a haploid human gamete. Chromosomal abnormalities include a total of three copies of the X chromosomes and four copies of chromosome 17 and chromosome 22. [7] [8] The presence of multiple X chromosomes and the lack of any trace of Y chromosome derived sequence suggest that the source fetus was female.

The 293T cell line was created in Michele Calos's lab at Stanford by stable transfection of the HEK 293 cell line with a plasmid encoding a temperature-sensitive mutant of the SV40 large T antigen; it was originally referred to as 293/tsA1609neo. [9] The first reference to the cell line as "293T" may be its use to create the BOSC23 packaging cell line for producing retroviral particles. [10]

Variants

Multiple variants of HEK 293 have been reported. [11] [12]

HEK 293T

The transfection used to create 293T (involving plasmid pRSV-1609) conferred neomycin/G418 resistance and expression of the tsA1609 allele of SV40 large T antigen; this allele is fully active at 33 °C (its permissive temperature), has substantial function at 37 °C, and is inactive at 40 °C. [14] 293T is very efficiently transfected with DNA (like its parent HEK 293). Due to the expression of SV40 large T antigen, transfected plasmid DNAs that carry the SV40 origin of replication can replicate in 293T and will transiently maintain a high copy number; this can greatly increase the amount of recombinant protein or retrovirus that can be produced from the cells.

The full genome sequences of three different isolates of 293T have been determined. They are quite similar to each other but show detectable divergence from the parental HEK 293 cell line. [15]

HEK293-ENT1KO

This mutant strain does not express of the equilibrative nucleoside transporter ENT1. The gene was knocked out using CRISPR-CAS9 and the cell line retains ENT2 expression. [16]

Applications

Immunofluorescent HEK 293 cells HEK 293.jpg
Immunofluorescent HEK 293 cells

HEK 293 cells are straightforward to grow in culture and to transfect. They have been used as hosts for gene expression. Typically, these experiments involve transfecting in a gene (or combination of genes) of interest, and then analyzing the expressed protein. The widespread use of this cell line is due to its transfectability by the various techniques, including calcium phosphate method, achieving efficiencies approaching 100%.

Examples of such experiments include:

HEK 293 cells were adapted to grow in suspension culture, as opposed to proliferation on plastic plates, in 1985. [22] This enabled the growth of large amounts of recombinant adenovirus vectors.

A more specific use of HEK 293 cells is in the propagation of adenoviral vectors. [23] Viruses offer an efficient means of delivering genes into cells, which they evolved to do, and are thus of great use as experimental tools. However, as pathogens, they also present a risk to the experimenter. This danger can be avoided by the use of viruses which lack key genes, and which are thus unable to replicate after entering a cell. In order to propagate such viral vectors, a cell line that expresses the missing genes is required. Since HEK 293 cells express a number of adenoviral genes, they can be used to propagate adenoviral vectors in which these genes (typically, E1 and E3) are deleted, such as AdEasy. [24] However, homologous recombination between the inserted cellular Ad5 sequence and the vector sequence, although rare, can restore the replication capacity to the vector. [25]

An important variant of this cell line is the 293T cell line. It contains the SV40 large T-antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of desired gene products. HEK 293, and especially HEK 293T, cells are commonly used for the production of various retroviral vectors. [26] Various retroviral packaging cell lines are also based on these cells.

Native proteins of interest

Depending on various conditions, the gene expression of HEK 293 cells may vary. The following proteins of interest (among many others) are commonly found in untreated HEK 293 cells:

Bioethics

Alvin Wong, a Catholic bioethicist, argues that despite the uncertainty over the origin of the embryonic cells used to obtain the cell line, one can infer that it came from a voluntary abortion. To some, this may present an ethical dilemma for using HEK 293 and derivative products, such as vaccines and many medications. [31] [32] [33] [34]

On 21 December 2020, the Roman Catholic Congregation for the Doctrine of the Faith stated that the moral duty to avoid vaccines made from cell lines derived from fetuses is, "not obligatory if there is grave danger, such as the otherwise uncontainable spread of a serious pathological event -- in this case, the pandemic spread of SARS-CoV-2 virus that causes Covid-19". The statement then justifies the use of other vaccines, "all vaccinations recognized as clinically safe and effective can be used in good conscience..." [35]

During the COVID-19 pandemic, anti-vaccination activists noted that HEK 293 cells are used in the manufacturing of the Oxford–AstraZeneca COVID-19 vaccine (AKA AZD1222). The cells are filtered out of the final products. [36]

Regeneron Pharmaceuticals, the maker of REGN-COV2, a therapeutic antibody cocktail used to alleviate symptoms of patients with COVID-19, did not use HEK 293T cells to produce the antibody cocktail but did use those cells to assess the potency of the drug. [37] [32]

In response to ethical concerns of vaccine production, several strategies for clinicians to discuss with their patients have been suggested. [38]

See also

Related Research Articles

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A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

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

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.

<i>Adenoviridae</i> Family of viruses

Adenoviruses are medium-sized, nonenveloped viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from their initial isolation from human adenoids in 1953.

<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.

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term, as the term "transformation" is also used to refer to a cell's progression to a cancerous state (carcinogenesis). Transduction is often used to describe virus-mediated gene transfer into prokaryotic cells.

<span class="mw-page-title-main">SV40</span> Species of virus

SV40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that is found to cause tumors in humans and animals, but most often persists as a latent infection. SV40 has been widely studied as a model eukaryotic virus, leading to many early discoveries in eukaryotic DNA replication and transcription.

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

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<span class="mw-page-title-main">Viral vector</span> Biotechnology to deliver genetic material into a cell

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<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.

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<span class="mw-page-title-main">H19 (gene)</span> Negative regulation (or limiting) of body weight and cell proliferation

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<span class="mw-page-title-main">BacMam</span> Use of baculovirus to deliver genes to mammalian cells

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<span class="mw-page-title-main">Immortalised cell line</span> Lineage of cells that evades senescence and continues dividing

An immortalised cell line is a population of cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells can therefore be grown for prolonged periods in vitro. The mutations required for immortality can occur naturally or be intentionally induced for experimental purposes. Immortal cell lines are a very important tool for research into the biochemistry and cell biology of multicellular organisms. Immortalised cell lines have also found uses in biotechnology.

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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">Lex van der Eb</span> Dutch molecular biologist and virologist

Alex Jan"Lex"van der Eb is a Dutch molecular biologist and virologist. He was a professor of fundamental tumor virology and later molecular carcinogenesis at Leiden University from 1979 to 2000. He has performed research in adenoviruses and was fundamental in the creation of the technique of calcium phosphate transfection and the founding of the HEK 293 and PER.C6 cell lines.

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References

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  2. 1 2 Austriaco N (May 25, 2020). "Moral Guidance on Using COVID-19 Vaccines Developed with Human Fetal Cell Lines". Public Discourse. Witherspoon Institute. Retrieved December 23, 2020. I received an e-mail a few months ago from Professor Frank Graham, who established this cell line. He tells me that to the best of his knowledge, the exact origin of the HEK293 fetal cells is unclear. They could have come from either a spontaneous miscarriage or an elective abortion.
  3. ↑ van der Eb A. "USA FDA CTR For Biologics Evaluation and Research Vaccines and Related Biological Products Advisory Committee Meeting" (PDF). Lines 14–22: USFDA. p. 81. Archived from the original (PDF) on 2017-05-16. Retrieved August 11, 2012.{{cite web}}: CS1 maint: location (link)
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  16. ↑ Shahid, Nayiar; Chromwell, Christopher; Hubbard, Basil; Hammond, James (May 2021). "Characterization of a Novel HEK293 Cell Line (HEK293-ENT1KO) to Assess the Role of Equilibrative Nucleoside Transporter Subtype-2". The FASEB Journal. 35 (S1). doi: 10.1096/fasebj.2021.35.S1.02185 . ISSN   0892-6638.
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  32. 1 2 Schorr I (20 December 2020). "The Facts about the COVID Vaccines and Fetal Cell Lines". National Review.
  33. ↑ "Balkan Insight: Tulpina religioasă a anti-vaccinismului se răspândește în Europa Centrală și de Est - International - HotNews.ro". 20 January 2021.
  34. ↑ RELIGIOUS STRAIN OF ANTI-VAX GROWS IN CEE, balkaninsight.com
  35. ↑ "Nota della Congregazione per la Dottrina della Fede sulla moralità dell'uso di alcuni vaccini anti-Covid-19". press.vatican.va. Retrieved 2024-03-06.
  36. ↑ Rahman G (26 November 2020). "There are no foetal cells in the AstraZeneca Covid-19 vaccine". Full Fact.
  37. ↑ Regalado, Antonio (7 October 2020). "Trump's antibody treatment was tested using cells originally derived from an abortion". MIT Technology Review. Retrieved 2021-09-30. It's how you want to parse it," says Alexandra Bowie, a Regeneron spokesperson. "But the 293T cell lines available today are not considered fetal tissue
  38. ↑ Zimmerman, Richard K. (2021-07-13). "Helping patients with ethical concerns about COVID-19 vaccines in light of fetal cell lines used in some COVID-19 vaccines". Vaccine. 39 (31): 4242–4244. doi:10.1016/j.vaccine.2021.06.027. ISSN   1873-2518. PMC   8205255 . PMID   34172329.
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