HEK-ing the 293 Cell Line

Edited by Eldrian Tho.

Human Embryonic Kidney Cells (abbreviated as HEK cells) are one of the most well-known cell lines in industrial laboratories. But what the HEK are they anyway? Let’s find out and see.

History of HEK cells

The typical cell line of HEK cells, HEK293, was initially isolated and grown by the Dutch biologist Alex van der Eb in the early 1970s in Leiden, Netherlands.¹ Frank Graham, a post-doc, then transfected these cells with adenoviral genes (specifically, adenovirus 5 DNA) in van der Eb’s laboratory. It was his 293rd experiment, hence the tag HEK 293.⁴

Graham performed the transfection process on the HEK cell about eight times, with only a single clone for a few months. After it presumably adapted to tissue culture, the cell finally developed to form the stable HEK 293 cell line.¹

What is the origin of the HEK 293 cell line?

For many years, HEK 293 cells were thought to be derived from a fibroblastic, endothelial or epithelial cell of the kidneys.¹ However, investigations have shown that this was not the case because Graham performed an adenovirus transfection on these cells, after which they were deemed inefficient. Therefore, this suggested that the HEK 293 cell lines came from a cell with a rather unusual feature.

Graham and his coworkers have proven that the cell lines generated by the adenovirus transformation have a common characteristic; they all had immature neurons. Therefore, this depicted that the adenovirus transformed a neuronal lineage cell in the culture of kidney cells.

Transcriptomes and genome studies have compared the HEK 293 transcriptomes with other main tissues in the kidney and concluded that they resembled the adrenal cells the most. This shows that adenoviruses are much more efficient at transforming neuronal lineage cells when compared to human kidney epithelial cells. Therefore, the origin of the HEK 293 cells has a great likelihood to have come from an embryonic adrenal precursor cell.

Fun Facts: Genome and Chromosome number of HEK cells

HEK 293 has a modal chromosome number of 64 and is described as hypotriploid, meaning it has less than three times the chromosome number of a haploid human gamete. They also have chromosomal abnormalities, such as three copies of X chromosomes and four copies of chromosomes 17 and 22. The absence of Y chromosomes has provided evidence that the source of the kidney cells was a female fetus.

What are the HEK 293 cells used for?

The corporation of adenoviral genes into HEK cell genomes has produced cells that are efficient at producing increased amounts of recombinant proteins from plasmid vectors, especially those carrying the CMV (transcription factor) promoter region.⁴ These cells lack the dangerous viral key genes, which allow them to be safe to handle.

Besides that, they are easy to grow in culture and to be transfected, which allows them to serve as hosts for gene expressions. Typically, experiments with HEK cells involve the transfection of a gene of interest to allow the analysis of the expressed protein. The results of the expressed protein can provide explanations for hypotheses revolving around the interaction between proteins, the effects of drugs etc.²

A remarkable example of the effectiveness in expressing novel proteins by this cell line would be the transfection of the cells with the recombinant activated protein C and later tested for the treatment of sepsis.

In fact, there are many features of the HEK 293 cells that are advantageous for gene therapy:

1. They are easy to grow

2. They are easy to maintain

3. They have a consistent and high reproducibility

But, to ensure the cells work at their best, they have to be living under the right conditions.

Care of HEK 293 cells

1. Growth medium

High-glucose growth media like Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) to a concentration of 10% should keep the HEK293 cells happy.³

*FBS helps to promote cell proliferation with growth factors and cell attachment to become confluent *

2. Growth conditions

HEK293 cells grow well in an incubator set to 37°C and in 5% of CO2 for gas exchange to take place

*When storing in the incubator, flasks should be ventilated to allow gas exchanges to occur

3. Storage conditions

HEK 293 cells should be stored in the liquid nitrogen vapour phase to ensure no loss in viability. When freezing, DMSO is added to prevent the ice crystals from puncturing the cells to avoid apoptosis. Ideally, an isopropanol storage box will allow gradual freezing of the cells at a rate of 1°C per minute before transferring them to the liquid nitrogen vapour phase.

4. Thawing

Cells will have to be thawed as soon as possible due to the toxicity of DMSO on the HEK 293 cells if exposed for a long period of time. They are supposed to be agitated in a 37°C water bath for 2 minutes before adding them to pre-warmed media (DMEM and FBS).

5. Culture Period

While the HEK 293 cells are deemed immortal, too much of everything is still bad for them. It is generally advised that the number of times the cells can be passaged (harvested and transferred to a new medium) is below 20 to avoid the negative consequences of downstream experiments.

Current Applications of HEK 293 cells

Receptor Signaling

• HEK 293 are used for expressing cell membrane receptors and ion channels.

• Studies have been using this cell line to investigate the signalling pathways of G protein-coupled receptors that are shown in diseases.

• The cells are also used to characterize receptors in organisms and elucidate the binding affinity of ligands on the receptors.

Protein Production

• A 2017 study has demonstrated the large-scale transfection of HEK 293SF cells to mass-produce H1N1 vaccines

• The cell line has also been manipulated to mass-produce therapeutic proteins like lysosomal acid lipase (LAL) and immunoglobulin G1 (lgG1)

Cancer Research

• HEK 293 cells are capable of forming tumours. In 2017, cells that express toll-like receptor 4 were used to elucidate the role of Metformin (type II diabetes medication) to treat cancer.

• It was found that Metformin was able to suppress the expression of inflammatory chemokine interleukin-8 (CXCL8) and cell migration, thus inhibiting the gradual evolution of cancer.

• It is also widely used as a control to test the effects of treatment on cancer-specific cell lines.

• A recent study has investigated the effects of the phytochemical compound thymoquinone (TQ) on breast cancer.

• The results have shown that TQ down-regulated the vascular endothelial growth factor, which is a regulator of tumour angiogenesis, as well as the up-regulation of tyrosine kinase receptors.

Are HEK 293 cells subjected to limitations?

Sadly, these cell lines need to be treated like royalty or else they will persecute you by destroying your experiment.

Its protocols largely revolve around avoiding any traces of contamination, so what kind of issues are there?

1. Bacterial contamination

If left unchecked, bacterial infections in a culture of HEK 293 cells will cause pH, colour and turbidity to change, making it inhabitable for the cells. Thus, they will not grow well. If you were to place a contaminated flask with bacteria under a microscope, there would be squiggly moving lines on a slate of cells, which is not ideal to proceed.

2. Viral contamination

This cell line is particularly susceptible to viral infections like most human cells. They are not as obvious as compared to bacterial contamination, so they will have to be detected through virus-specific PCR- based assays.

In short, do handle these cells as if your life depends on them.

The Variants of HEK 293 cell lines

Here is an image of the various cell lines that exist in laboratories worldwide now with a specificity according to the antigens present.

Derivative⁴	Features
​293-F	​Fast growing; grow in suspension; grow in serum-free media.
293-H	Fast growing; grow in suspension; grow in serum-free media; good adherence in plaque assays.
293T	High transfectability; contains the SV40 T-antigen.
293FT	Derivative of the 293-F cell line; fast growing; contains the SV40 T-antigen.
293E, also known as 293-EBNA1	Expresses the EBNA-1 protein.

In conclusion, HEK 293 cells rule the world (at least, about to)

If you are planning to join the field of genome engineering or biotechnology, these cell lines will be something you may come across. With its vast uses, it will definitely be a promising starting material in the field of cancer research in the future.

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Glossary

1. Cell line - a cell culture selected for uniformity from a cell population derived from a usually homogeneous tissue source (such as an organ)

2. Adenoviral gene - One of a number of genetically-engineered adenoviruses designed to insert a gene of interest into a eukaryotic cell where the gene of interest is subsequently expressed.

3. Transfection - process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.

4. Transcriptomes - the sum total of all the messenger RNA molecules expressed from the genes of an organism

5. Therapeutic proteins - medicines that are genetically engineered versions of naturally occurring human proteins

6. Transfectability - Ability to be transfected

7. Neuronal lineage - Neurons descended from their progenitors, aka their descendants

8. Precursor - a substance from which another is formed, especially by metabolic reaction.

9. Haploid - a cell or nucleus having a single set of unpaired chromosomes.

10. Plasmid vector - a type of vector commonly used in recombinant DNA technology. It is a double-stranded, circular, extrachromosomal DNA that is separated from the genomic DNA. Also, it has the remarkable property of self-replication inside the host cell.

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References:

1. Wikipedia, the free encyclopedia - HEK 293 cells https://en.wikipedia.org/wiki/HEK_293_cells

2. HEK 293 cells: Applications and Advantages | News Medical & Life Sciences, 2019 https://www.news-medical.net/life-sciences/HEK293-Cells-Applications-and-Advantages.aspx

3. HEK 293 cells: Background, Applications, Protocols, and More | Synthego https://www.synthego.com/hek293

4. What the HEK? A Beginner’s Guide to HEK 293 Cells | BiteSizeBio, 2021 https://bitesizebio.com/45489/guide-to-hek293-cells/