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HEK293T Cells: Unlocking the Potential in Transfection Studies

HEK293T cells are human embryonic kidney cells widely applied in industrial biotechnology, toxicology, and cancer research. These immortalized cells are also used to produce a variety of therapeutic proteins and viruses.

General characteristics and origin of HEK293T cells

This article section will cover the origin and general information about the HEK293T cell line. 

  • The HEK293T cell line originated from primary human embryonic kidney cells cultured in the laboratory. Researchers developed these cells in the early 1970s by transfecting embryonic kidney cells with sheared adenovirus type 5 DNA fragments. Researchers established the HEK293T cell line by introducing the simian virus 40 (SV40) large T-antigen to the genome of HEK293 cells. This modification enabled researchers to easily transfect 293 cells and made them suitable for protein production and gene expression studies [1].
  • HEK293T cells possess an epithelial-like morphology. They represent an elongated and flattened shape with a definite cell boundary.
  • The size of HEK293T cells ranges between 11 and 15 µm in diameter.
  • The SV40 large-t antigen HEK293T cells possess a complex karyotype. These cells are hypotriploid, containing 3 times fewer chromosomes than a haploid gamete, and they possess a modal chromosome number of 64.

What is the difference between HEK293T and HEK293?

Both HEK293 and HEK293T cell lines are of human origin. HEK293T is a popular derivative of the HEK293 cell line. Scientists developed these cells from the original human embryonic kidney 293 cells by transfecting them with SV40 large T antigen, while they established the immortalized HEK293 cells by transforming and culturing human embryonic kidney cells with sheared human adenovirus 5 DNA fragments.

Human embryonic kidney 293 cells observed under the microscope.

Cell Technologies and Biomedical Applications of HEK293T cells

Cell Culture and Banking in HEK293T Research

HEK293T cells, which are derived from human embryonic kidney 293 cells, find extensive use in cell culture because of their robust growth and ease of transfection. To work with these cells, researchers must prioritize cell banking, which involves storing cells for extended research and therapeutic use. They need to use a graded cell banking approach to preserve cell properties and ensure long-term cell viability. The generation of cell banks requires adherence to good manufacturing practice regulations to ensure the viability and integrity of cells for therapeutic applications.

Good manufacturing practices are crucial in the production of HEK293T cell banks, which are foundational for both research and therapeutic applications. The master cell bank serves as a reference point for all subsequent cellular products. The manufacturing of these cells for therapeutic applications, such as lentiviral production for gene therapies, follows strict regulatory standards to ensure the safety and efficacy of the end products.

Protocols and Assays Using HEK293T

In cytotechnology, specific protocols and assays are designed to evaluate the HEK293T cell properties. These include assessing the efficacy of gene therapy vectors and the cell's interaction with the extracellular matrix in a dish or a suspension culture. To maintain the integrity of the HEK293T cells, the researchers choose reagents for transfection with precision and subject raw materials to stringent quality control tests.

Research applications of the HEK293T cell line

  • Vaccine development: The HEK293T cell line has been used to study viruses and produce viral vector-based vaccines to combat a variety of viral infections. A study used this embryonic kidney cell line to investigate the structural and functional basis of COVID-19 virus cell entry through human angiotensin-converting enzyme 2 (ACE2) [3]. In addition, a recent study used HEK93T cells to generate SARS-CoV-2 spike-pseudotyped lentivirus particles [4].
  • Toxicology research: This human embryonic kidney cell line is widely used to test the toxicity and efficacy of drugs. Research conducted in 2022 used HEK293T as a normal human cell line to validate the cytotoxic potential of Caladium lindenii extracts against the liver cancer cell line HepG2 [5].
  • Gene expression studies: The SV40 large T antigen HEK293T cell line is highly amenable to transfection, thus suitable for gene expression studies. A study used HEK293T cells to study the role of the long noncoding RNA SNHG16 in regulating trophoblast functions. The study revealed that LncRNA SNHG16 interacts with the miR-218-5p/LASP1 axis to mediate these effects [6].

Cell Therapy Innovations Using HEK293T Cells

Advancements in Cell Therapy with HEK293T

HEK293T cells contribute significantly to the cell therapy landscape, especially in the generation of viral vectors for gene therapy. These cells are essential in manufacturing processes that adhere to good manufacturing practice regulations, as they ensure the production of high-quality gene therapy products. The training of manufacturing personnel also focuses on handling the unique properties of HEK293T cells and maintaining a high standard for the medicinal products derived from these cells.

HEK293T Cells in Clinical Trials and Gene Therapy

The HEK293T cell line plays a pivotal role in the development of gene therapy products and is integral to clinical trials aiming to bring new cell therapies to the market. This involves leveraging the cell line's high transfectivity for gene delivery, utilizing vectors such as lentiviral packaging vectors, where the integrase D64V mutation has been a notable advancement for increasing safety.

Innovative Techniques in HEK293T Cell Culture

The versatility of HEK293T cells supports innovative techniques in both two-dimensional and more complex-dimensional cell cultures. This adaptability is key in exploring cellular products for various types of biomedical research, including cancer research, where these cells are used to study tumorigenic processes and drug testing. Additionally, the HEK293T line is instrumental in the production of lentiviral particles, which are critical for both research and therapeutic vector manufacturing processes.

Purchase HEK293 Cells for Your Research

Elevate your research with our HEK293 cells, renowned for their adaptability in gene expression studies and vaccine production. Our offerings include versatile derivatives such as HEK293T, the HEK293 suspension-adapted line, HEK293T/17, AAV-293, and 2V6.11. Explore our extensive selection to support and advance your experimental work.

HEK293 Cells
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Culturing information for HEK293T cell line

HEK293T cells are extensively cultured in research laboratories. Before developing HEK293T cell culture, you must know: What is the doubling time of HEK293T cells? What is HEK293T media?  What is the HEK293T seeding density?

Key Points for Culturing HEK293T Cells

Doubling Time:

The doubling time reported for HEK293T cells is 30 hours.

Adherent or in Suspension:

HEK293T is an adherent cell line.

Seeding Density:

HEK293T cells are seeded at a density of 1 x 104 cells/cm2. At this seeding density, cells can make a confluent monolayer in approximately 4 days. For seeding, adherent cells are detached by using the accutase dissociation solution. Detached cells are centrifuged and then carefully resuspended using a growth medium. Afterward, cells are dispensed into new flasks for culturing.

Growth Medium:

HEK293T cells are cultured in EMEM (Eagle's minimal essential medium) containing 1.0 g/L L-glucose, 2.2 g/L NaHCO3, 2.0 mM L-glutamine, and 10% fetal bovine serum. Medium should be replaced twice a week.

Growth Conditions:

HEK293T cell cultures are kept in a 37°C humidified incubator with a 5% CO2 supply.

Storage:

Human embryonic kidney HEK293T cells are stored in the vapor phase of liquid nitrogen or ata temperature a temperature below -150°C for longer.

Freezing Process and Medium:

HEK293 cells can be frozen in CM-1 or CM-ACF freezing media. Slow freezing processes that allow a gradual 1°C drop in temperature are recommended for HEK293T cells to protect their viability.

Thawing Process:

A frozen cell vial is rapidly agitated in a water bath (37°C) until a small ice clump is left. Cells are resuspended in media and centrifuged to eliminate components of freezing media. Recovered cells are then cultured in new flasks containing growth medium. 

Biosafety Level:

Biosafety level 1 laboratory is required to handle and maintain HEK293T cell cultures.

 

Semiconfluent and confluent monolayers of HEK293T cells.

HEK293T cell line: Advantages & Limitations

A unique combination of advantages and limitations is associated with human embryonic kidney 293T cells. Here, we will go over a few main advantages and disadvantages of this cell line.

Pros and Cons of HEK293T cells

The significant advantages of HEK293T cells include their high transfectivity, where the cell line exhibits remarkable efficiency in taking up foreign DNA and producing abundant proteins. This quality makes them widely favored for both transient and stable transfection studies. Additionally, HEK293T cell cultures are known for their ease of maintenance, making them an excellent choice for various laboratory experiments due to their robustness and straightforward handling requirements.

However, there are limitations associated with HEK293T cell cultures. One of the primary concerns is the risk of microbial contamination, which can significantly impact cell morphology, gene expression, and other critical characteristics, potentially leading to inaccurate experimental results. Furthermore, while HEK293T cells are suitable for long-term experiments, extended culture periods may compromise the cells' health. This can affect their transfection efficiency and growth rates, which is why it is generally recommended to limit their passage number to 20 or below to maintain cell integrity.

Frequently Asked Questions About HEK293T Cell Culture and Applications

Adherent culture refers to the method by which HEK293T cells attach to a surface, such as a dish, for growth. This method is crucial for maintaining the different morphologies and cellular behaviors observed in these cells. Materials like polystyrene or glass dishes coated with substances like gelatin are often used to enhance cell attachment and growth.
HEK293T cells are cultured and expanded under strict laboratory conditions to create a master cell bank. This bank serves as a consistent cell source for further research and manufacturing purposes. Process validation and quality control tests are critical steps to ensure that each batch of cells is viable and uncontaminated.
Essential materials for culturing HEK293T cells include culture dishes or flasks, culture media, and reagents such as enzymes and buffers. Peptides, proteins, and enzymes may also be designed and added to the culture to study their effects on the cells or to produce specific cellular responses.
Yes, HEK293T cells are frequently used as packaging cells to produce lentiviral particles. They are preferred for their high transfection efficiency and ability to produce high viral titers, which is crucial in gene therapy applications and the creation of cell therapy products.
Quality control measures for HEK293T cells include performing simulation tests to mimic the final product's environment, testing for contaminants such as air particles and pathogens, and ensuring cell viability and genetic stability. These tests guarantee that the cells are safe and effective for therapeutic use.
HEK293T cells are instrumental in renal cancer research due to their ability to be genetically manipulated to express renal cancer cell properties or to be co-cultured with renal cancer stem cells. This allows for the study of cancer cell behaviors in tissues, drug testing, and the investigation of potential treatments.
HEK293 and HEK293T cells are both derived from human embryonic kidney cells but have some key differences. The main difference is that HEK293T cells have been genetically modified to express the SV40 large T antigen, which enhances the cells' transfection efficiency. This T antigen allows for episomal replication of plasmids containing the SV40 origin of replication, making HEK293T cells highly suitable for transient transfection and viral production.
HEK293 cells are used in a wide range of biomedical research applications. Their versatility makes them suitable for studies in gene expression, protein production, and even in the development of viral vectors for gene therapy and vaccines. Their easy culture and maintenance in the laboratory further contribute to their popularity in research settings.
HEK293T cells are particularly used for transfection due to the presence of the SV40 large T antigen, which significantly improves their ability to take up and express foreign DNA. This makes them an excellent tool for studying gene function, protein interactions, and for the production of recombinant proteins and lentiviral vectors.
The "T" in HEK293T denotes the presence of the SV40 large T antigen within these cells. This antigen disrupts the function of tumor suppressor proteins in the cell, allowing for easier insertion and expression of foreign DNA, thus enhancing the cell line's utility in various transfection and gene expression studies.

HEK293T cells: Research publications

In this section, we have mentioned a few promising research publications featuring HEK293T cells.

Development of SARS-CoV-2 mRNA vaccines encoding spike N-terminal and receptor binding domains

This publication will be published in BioRxiv in 2022. This study used HEK293T cells to develop COVID-19 virus mRNA vaccines encoding spike gene N-terminal and RBD (receptor binding domain) domains.

Circular HER2 RNA positive triple negative breast cancer is sensitive to Pertuzumab

This research is published in Molecular Cancer in 2020. The study proposed that circular HER2 RNA expression in triple-negative breast cancer cells makes it sensitive to Pertuzumab drug treatment. Researchers used HEK293 cells for lentivirus production and transfection of the circular HER2 gene in this study.

Antiviral role of IFITM3 in prototype foamy virus infection

This paper was published in the Virology Journal in 2022. This study utilized HEK293T cells to investigate the antiviral effect of IFITM3 (interferon-induced transmembrane protein 3) in a prototype foamy virus (PFV) infection.

MiRNA-21 mediates the antiangiogenic activity of metformin through targeting PTEN and SMAD7 expression and PI3K/AKT pathway

This research paper in Nature Scientific Reports (2017) used HEK293T cells and studied that miRNA-21 mediates metformin-induced antiangiogenic effects by regulating the PI3K/AKT signaling pathway and SMAD7 and PTEN gene expression.

MicroRNA-608 inhibits proliferation of bladder cancer via AKT/FOXO3a signaling pathway

This research was published in the Molecular Cancer journal in 2017. This study used HEK293 cells to investigate the antiproliferative potential of miRNA-608 against bladder cancer.

Resources for HEK293T cell line: Protocols, Videos, and More

The following are a few resources on HEK293T cells::.

Cell culture protocols

The cell culture protocols for HEK293T cells are listed here.

References

  1. Tan, E., et al., HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Frontiers in Bioengineering and Biotechnology, 2021, 9.
  2. Kim, M.J., et al., AMPKα1 Regulates Lung and Breast Cancer Progression by Regulating TLR4-Mediated TRAF6-BECN1 Signaling Axis. Cancers (Basel), 2020, 12(11).
  3. Wang, Q., et al., Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell, 2020. 181(4): p. 894–904.
  4. Gale, E.C., et al., Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2. bioRxiv, 2021.
  5. Kalsoom, A., et al., In Vitro Evaluation of Cytotoxic Potential of Caladium lindenii Extracts on Human Hepatocarcinoma HepG2 and Normal HEK293T Cell Lines. Biomed Res Int, 2022, p. 1279961.
  6. Yu, Z., et al., LncRNA SNHG16 regulates trophoblast functions by the miR-218-5p/LASP1 axis. J Mol Histol, 2021. 52(5): p. 1021-1033.

 

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