HEK293 Cells: A Cornerstone in Modern Cellular Research and Biotechnology

Human embryonic kidney 293 (HEK293) cells are a human embryonic kidney cell line that has gained widespread popularity in the scientific community because of their versatility and usefulness in a wide range of research applications. The cell line was established in the early 1970s and has since been used for vaccine development, cancer research, drug testing, and signal transduction. This blog post will explore all aspects of the HEK293 cell line, including its origin, culture information, advantages and drawbacks, applications, and resources.

HEK293 Cells: General Information and Origin

What are HEK293 cells?

HEK293 cells are a human embryonic kidney cell line derived from the kidney tissue of an electively terminated human embryo of unknown parenthood. The cells were established by a Dutch biologist named Alex Van der Eb in the early 1970s. They were later immortalized through a transformation with a truncated adenovirus 5 by researcher Frank Graham.

Initially, the cells seemed challenging to transform. However, after many continuous efforts, cell growth happened from an isolated single transformed clone [1]. The cell's transfection with adenovirus 5 led to the inclusion of the E1A and E1B genes in the cell's genome, which prevents cell death and allows for abundant protein production. Before immortalization, the fetal kidney cells were not adequately characterized, so their exact cell type is unknown.

Embryonic kidneys are composed of endothelial, epithelial, and fibroblast cells, so HEK 293 cells likely belong to these cells. However, the mRNA and gene products suggest that they are neuronal cells. It is possible that the addition of Ad5 altered the cellular phenotype and gene expression. Fun fact: The "293" in HEK293 refers to the 293rd experiment performed by Graham.

Fun fact: The "293" in HEK293 refers to the 293rd experiment performed by Graham.

Characteristics of HEK293 cells

• Morphology
• Cell size
• Genome and ploidy (chromosome number)

HEK293 cells have a shape that resembles epithelial cells. Embryonic kidneys are composed mainly of fibroblast, endothelial, and epithelial cells. Thus 293 cells resemble one of these cell types in shape.

The size of HEK 293 cells falls between 11 to 15 µm, which can be affected by the culture conditions. In culture, the cells may appear flattened when grown on a surface or rounded in suspension. HEK293 cells are hypotriploid, and about 30% of HEK293 cells have a modal ploidy of 64 chromosomes, but some cells have even more chromosomes. The cells also have three copies of the X chromosome and a 4-kilobase pair fragment of adenovirus 5 integrated into chromosome 19.

Comparison of HEK293 Vs. HEK293T cell line

Many derivatives have been derived from the parental HEK 293 cells, such as the common 293 cell derivatives HEK293T and HEK293F cells. HEK293T cells are one of the most widely used derivatives and were created by incorporating a temperature-sensitive SV40 T-antigen mutant into the original HEK 293 cell genome. Expression of the T-antigen allows for the replication of plasmids with an SV40 origin of replication when transfected into 293-T cells, leading to increased production of recombinant proteins [2]. For more information on HEK cell line derivatives, including their development and characteristics, refer to this review article.

Basics of HEK293 Cell Culturing: A Step-by-Step Guide

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The HEK293 Cell Line in Research & Industry

The applications of HEK293 cells are diverse and significant. They are frequently used as a system for the expression and production of recombinant proteins. Due to their human origin, proteins produced in these cells are more likely to be similar to their natural human counterparts in terms of structure and function, which is crucial for therapeutic applications.

Moreover, HEK293 cells are often employed in the study of gene function and regulation, as they readily take up foreign DNA, making them an excellent model for genetic manipulation. These cells also play a critical role in the production of adenoviral vectors, which are used in gene therapy and vaccine development, including the rapid generation of vaccines for COVID-19.

Vaccine and Protein production: HEK 293 cells are suitable for large-scale protein and therapeutic vaccine manufacture. The cell line is further used to generate viral vectors such as adeno associated and adenoviral vectors. Recently, HEK293 cells have been used for producing a crucial recombinant protein, erythropoietin (EPO).

Drug testing: HEK293 cells are frequently used for testing the toxicity of drugs and natural products.

Cancer research: 293 cells are tumorigenic, and crucial gene expression changes can aggravate tumorigenesis in this cell line. Therefore, the 293 cell line is frequently used in cancer studies for understanding the underlying molecular mechanisms and drug development.

Transfection studies: Transfection is the process of introducing nucleic acids into cells, and HEK293 cells are particularly amenable to this process. More on this topic is explained below.

The Role of HEK293 in Vaccine and Protein Production

In vaccine production, HEK293 cells have been instrumental in the development of adenovirus-based vaccines. Their ability to grow in suspension cultures allows for scalable processes, pivotal in meeting global vaccine demands. Additionally, their human origin provides an advantage over other cell lines, as they can perform human-like post-translational modifications, ensuring the biological efficacy of the produced vaccines.

The versatility of HEK293 cells extends to the production of complex proteins, including monoclonal antibodies and biosimilars, which are used in the treatment of cancer, autoimmune diseases, and other conditions. Their capacity to accurately fold and modify proteins makes them a preferred choice in the recombinant protein production industry.

Why Are HEK293 Cells Used for Transfection?

Transfection is the process of introducing nucleic acids into cells, and HEK293 cells are particularly amenable to this process. There are several reasons why HEK293 cells are favored for transfection:

1. High Transfection Efficiency: HEK293 cells have a high uptake rate for foreign DNA, which can be attributed to their ability to express certain viral genes that facilitate the entry of DNA into the cell.
2. Robust Growth: These cells grow rapidly and are relatively easy to maintain, which is beneficial for experiments that require quick and reliable results.
3. Adaptability: HEK293 cells can be grown in a variety of conditions, including adherent or suspension cultures, which makes them suitable for large-scale protein production.
4. Human Cell Line: As a human cell line, they provide more relevant biological context for human biology, which is particularly important in therapeutic research where the response in human cells is predictive of in vivo outcomes.
5. Versatility: They are able to produce proteins with complex post-translational modifications, a feature that is essential for the functionality of many proteins, especially therapeutic antibodies. 

HEK293 Subculture Protocol

Required Reagents

1. 1X Phosphate-Buffered Saline (PBS)
2. 10% Trypsin-PBS
3. Dulbecco's Modified Eagle's Medium (DMEM)

Procedure

Cell Preparation

1. Check the HEK cells under a microscope to ensure they are about 90% confluent.
2. Clean the workstation using aseptic techniques and sterilize the fume hood with UV light.
3. Wipe the workspace with 70% ethanol.
4. Pre-warm all reagents in a 37°C water bath.

Calculating Split Fraction and Seed Amount

1. Determine the split fraction, typically between 1:5 to 1:20.
2. Calculate the volume for pipetting using the formula: Vp = (S)(Vd).

Media Volumes and Splitting Protocols

For cell culture, different vessels require specific media volumes and have unique growth areas. For example, a 6-well plate has a growth area of 4.67 cm^2 per well and needs about 2.5 mL of media, whereas a 100 mm plate has a 55 cm^2 growth area, requiring 10 mL of media. The process of splitting cells involves removing old media, washing with PBS, incubating with Accutase, neutralizing with DMEM, centrifuging, resuspending in new media, and then seeding onto a new plate. For detailed steps and ratios for other vessels like 100 cm^2 flasks and 150 mm plates, please refer to the original source.

Frequently Asked Questions About HEK293 Cells

HEK293 cells are a subject of widespread use in scientific research, which naturally brings up numerous questions about their nature, origin, and characteristics. Below, we explore some of these common inquiries.