What is Transfection? A Practical Guide

Researchers use several terms to describe the transfer of nucleic acid into living cells or organisms, depending on the context. For example:

• Transfection is used to describe the transfer of nucleic acids, typically by non-viral methods, into animal cells.
• Transformation is often used for the same process in the context of plant, fungal, and bacterial cells. However, this term is typically not used in animal cell biology to avoid confusion with the term "malignant transformation" in cancer biology.
• Transduction refers to the viral-meditated transfer of nucleic acids into cells.

Transfection has a wide variety of applications in research and biological product manufacturing.

Scientists use cell culture transfection as part of a wide variety of experiments and procedures in the research lab. For example, they may introduce a newly discovered gene into cells to understand how its protein product functions or "knock out" a gene to study how cells behave in its absence. Another example includes inserting fluorescent proteins into mammalian cells so that they can be more easily observed using imaging devices.

Transfection plays multiple roles in gene therapy and regenerative medicine. One major role is in the manufacture of viral vectors, which are often used for in vitro delivery on genetic material into cells, such as in CAR T-cell therapy. In CAR T-cell therapy, patient-derived T-cells are genetically modified in vitro with viral vectors produced through transfection, and then returned to the body to target and destroy cancer cells. Transfection therefore indirectly supports these types of cell therapy applications.

Transfection also plays direct roles in the manufacture of cell therapy products intended for regenerative medicine or other therapeutic purposes. For example, an article in the Journal of Controlled Release notes that stem cells may be genetically modified using various transfection methods to improve their potential therapeutic activity or increase their survival.

According to an article in BMC Molecular and Cell Biology, transfection can be achieved via several basic methods:

1. Physical Transfection

Physical transfection methods deliver DNA or RNA into the cytoplasm or cell nucleus via physical disruption of the cellular membranes. Examples of physical transfection include biolistic particle delivery (also known as the gene gun method), direct microinjection, electroporation, and laser-mediated transfection.

Physical methods can have very high transfection efficacy but can be damaging to cells and even trigger significant cell death. In addition, most physical transfection methods require specialized equipment, and many have a low throughput.

2. Chemical Transfection

Chemical transfection methods rely on carrier molecules that encapsulate or otherwise bring the DNA or RNA into the cell. These carrier molecules include cationic lipid-based carriers, calcium phosphate, and cationic polymers.

Some chemical transfection methods, like calcium phosphate transfection, are inexpensive but have lower efficacy and a higher risk of damaging cells. These are best used with hardy, non-primary cell lines. Others, like cationic lipid carriers, are known for their high transfection efficacy and applicability to many cell types, but they can be more costly and may require more complex and optimized procedures.

3. Viral Transduction

Sometimes considered "biological transfection", viral-mediated transfer of genetic material—transduction—utilizes genetically engineered viruses such as adenoviruses, adeno-associated viruses or retroviruses to introduce DNA or RNA into cells. These viral vectors are typically produced by the other transfection methods.

Viral transduction is often preferred for its high efficiency and ability to achieve stable gene delivery. It may succeed with cells that do not yield easily to other transfection methods, although it is more technically challenging. Use of viral vectors carries risk of cellular immune response, potential mutation in host cells, and biosafety concerns, as well.

Stable and Transient Transfection Techniques

The nucleic acids inserted into cells during transfection can exist in either a stable or transient form. Physical, chemical, and biological transfection methods can each be used to achieve stable or transient transfection.

Transient transfection: With transient transfection, intersected nucleic materials (e.g., (e.g., plasmids, oligonucleotides, messenger RNA) do not merge with the host cell genome and will persist only for a limited time. Transient transfection is often simpler to achieve, but its use is limited to short-term procedures, such as gene silencing experiments or small-scale protein production. Physical and chemical transfection methods are typically used for transient transfection but can be optimized to provide stable transfection in certain cases.

Stable transfection: If you need to generate cells that will retain the inserted genetic material even after replication, choose a stable transfection technique in which the inserted genes either integrate into the cell genome or are carried on a persistent episomal vector. The genetic material of interest often carries a selection marker, such as an antibiotic resistance, to facilitate the identification and enrichment of stably transfected cells. Achieving stable transfection tends to be more time-consuming, particularly if using physical or chemical methods of DNA transfer. For this reason, biological methods, such as viral transduction, are often preferred because lentiviral or other retroviral vectors can efficiently integrate genetic material into the host genome, ensuring long-term expression.

To choose between transfection methods and between stable and transient transfection, consider the cell line you are using and which transfection methods have a track record of success with that cell line. Additionally, assess whether the cells of interest are primary, an immortalized cell line, or stem-like, as some cells are more sensitive to certain transfection methods.

Also consider the size and form of nucleic acid to be inserted (e.g., large plasmid, oligonucleotides, small RNA) and choose a technique that works well for that form.

Consider the downstream application of your cells as well. Some methods carry a higher chance of damaging sensitive cells, such as those used in cell and gene therapy. For instance, techniques that minimize cell stress, such as lipid-based transfection, may be necessary for therapeutic applications. For processes that will need to be scaled up, the overall cost and scalability of the transfection method are important considerations. For example, researchers working on AAV-based gene therapy programs are advised to consider the cell line they are using and their needs for scale-up flexibility when choosing between different transfection methods and cell growth platforms.

Every cell line and experimental procedure is different, and any transfection procedure may need to be optimized. Cell line health and viability, nucleic acid concentration, transfection reagent concentration, cell confluency, and procedure duration can all affect successful transfection. Here are a few other things to consider:

• Ensure cells are healthy and transfection reagents are stored properly to avoid loss of efficacy.
• Optimize reagent-to-nucleic acid ratios for your specific cell type, as overloading some transfection reagents can lead to cytotoxicity.
• Nucleic acid quality is another important factor. Genetic material with higher purity will typically deliver better transfection outcomes.
• Timing is key. Check your protocol or the reagent packaging for the correct incubation period. After transfection, cells are often stressed and need gentle handling to help them recover. Follow the reagent manufacturer's directions or your protocol for the recommended timing of media changes after transfection.

Using a tested protocol and proven reagents can improve your chances of success. Corning offers tested protocols for successful transfection. Collaborating with reagent manufacturers or consulting published studies can also inform protocol optimization for specific cell types or experimental goals. In addition, reagents, such as Corning^® transfectagro™ reduced-serum medium support efficient transfection, and Corning^® BioCoat^® gelatin plates support the growth of transfected cells.

Despite the popularity of methods like electroporation, lipofection, and viral transfection, challenges remain. Some cell types remain difficult to transfect. Large plasmids are often needed to deliver complex genetic machinery for applications such as CRISPR/Cas9 gene editing, but can be difficult to introduce into cells. In addition, tradeoffs between transfection efficiency and cell viability can cause delays in research and higher costs in cell therapy manufacturing.

To overcome these challenges, scientists are developing new transfection techniques for research and cell and gene therapy manufacturing. An article in Scientific Reports reveals how a research team used sudden depressurization to successfully transfect a DNA-carrying plasmid into embryonic stem cells, which can be difficult to transfect using conventional methods. The new technique, termed pressure-jump-poration, is similar to electroporation in that it temporarily increases the permeability of the cell membrane to nucleic material. However, pressure-jump-poration appears to work better for primary cell lines and to present less risk of DNA damage compared to electroporation.

Another paper published in Science Advances reports successfully using an acoustothermal method to permeabilize and transfect mesenchymal stem cells. This novel approach combines sound waves and heat to temporarily increase membrane permeability, enabling the delivery of large plasmids with reduced cytotoxicity. The researchers developed this technique to improve the transfection of large plasmids into stem cells while supporting transfection efficiency and cell viability.

Corning has decades of experience supporting cutting-edge innovations in cell culture and advanced therapies, both in academic and commercial labs. Our experts can help you identify the transfection techniques and products best suited to your particular application.

Learn more at 3D culture resources and cell and gene therapy resources with Corning Life Sciences.

Cell line health and viability, nucleic acid quality, transfection reagent, transfection duration, and serum affect successful transfection. Cells must be uncontaminated, grown in a fresh medium, and kept in appropriate incubation conditions. Check transfection protocols for serum requirements, too, because some require serum-free conditions.

Transfect cells at 40–80% confluency. Too few cells can cause poor growth due to limited cell-to-cell contact, while too many cells can lead to a resistance in uptaking foreign nucleic acid.

Most chemical transfection reagents also have optimal time windows between 5 and 30 minutes, depending on reagent. The optimal time depends on cell line, transfection reagent, and nucleic acid.

No. The interactions of every cell type's specific properties and every transfection reagent are complicated. Consequently, any efforts to predict transfection efficiency are wasted. However, some empirical values show various cell types, such as primary cells, quiescent cells, and suspension cells, are difficult to transfect.

Genetic material with higher purity will routinely deliver better transfection outcomes. Identifying contamination by lipopolysaccharides (endotoxins) that are introduced by bacteria during the manufacturing process is critical. The innate immune system can detect even trace amounts, significantly impeding the transfection process. It is recommended to use commercial kits to remove endotoxins from genetic material.

Plasmid cleaning based on miniprep protocols is not recommended because:

• Promotors have unique expression rates.
• Certain gene characteristics may heavily affect cell physiology or trigger cell death.
• The genetic material's dimensions and tertiary structure also impact transfection success.

Transfection is a fundamental part of gene therapy and regenerative medicine. Continued work with stem cells and 3D cultures is vital to further the progression of precision and personalized medicine.

Learn more about 3D cell culture with Corning Life Sciences.