Transfection Methods: A Primer

Among the many ways of introducing foreign nucleic acids into cells, some methods tend to predominate, especially when it comes to particular cell types. This is just as well, as cells can be finicky, with some common cell types gaining reputations for being amenable or incompatible with particular transfection methods and cargo types. In addition, each transfection method has distinct advantages and disadvantages depending on the researcher’s experimental goals. This article reviews common transfection methods, and compatibility with cell types and applications.

Chemical transfection methods

Common chemical transfection methods include DEAE-dextran, calcium phosphate co-immunoprecipitation, cationic lipids/liposomal reagents, and transfection using magnetic beads or particles. Most chemical methods are convenient, easy to learn, compatible with many cell lines, and don’t require special equipment. “Chemical transfection has come a long way, so most all commonly used cell lines are compatible with chemical transfection—the trick is finding the right formulation to use that balances efficiency and toxicity,” says Sandy Tseng, Technical Support Scientist at Mirus Bio.

"Chemical transfection is usually a first choice among customers due to its simplicity, cost, and quick turn-around time," according to Joanna Megdadi, Global Product Manager, Marketing, Gene Expression Business at Bio-Rad Laboratories. She notes that customers often use multiple delivery methods in their work depending on the molecule and cell type selected, but chemical transfection usually prevails. “However, chemical delivery is often less effective with hard-to-transfect cell types, such as primary blood cells, as well as cell types sensitive to chemical toxicity,” she says. “Delivery efficiency may also be compromised as molecule size increases, and is less successful with delivering ribonucleoproteins (RNPs), such as CRISPR/Cas9.”

Physical transfection methods

Physical methods—including microinjection, biolistic delivery, and electroporation—are especially useful for cell types that are difficult to transfect with other methods. Microinjection, in which cells are individually injected with nucleic acids, generally has high success rates, but the manual procedure necessarily limits the overall number of transfected cells. In biolistic methods, nucleic acids are affixed to inert particles, which are then shot into cells using a gene gun. Although this method is also limited in throughput, it is promising for in vivo applications because it can transfect cells deep within tissues.

Compared to microinjection and biolistics, electroporation can transfect more cells simultaneously. “It quickly enables molecule delivery through transient pores in the cell membrane without the need to target specific cell proteins, or the limitation of cell tropism,” says Megdadi. Compared to other transfection methods, electroporation works with almost any cell type, and can handle a wide range of cargo sizes. “It is also an excellent approach for CRISPR/Cas9 gene-editing workflows requiring mRNAs or RNP transfection into cells, without the need for transcription and translation steps necessary with viral CRISPR delivery,” adds Megdadi.

Lonza’s Nucleofector^® Technology is an electroporation-based method using specific solutions and optimized pulses that result in higher transfection efficiencies, even in difficult-to-transfect cells. Although the method is as convenient as chemical methods using transfection reagents, an important difference is that cell proliferation is not required for Nucleofection-based transfection. “Because Nucleofector^® Technology is independent of cell proliferation, it allows for efficient transfection of even non-dividing primary cells (like resting T cells or neurons) and difficult-to-transfect cell types,” says Andrea Toell, Senior Product Manager for Nucleofector Technology at Lonza Bioscience. “Compared to classical electroporation, it achieves much higher viabilities for primary cells and higher efficiencies with less amount of DNA.”

Nucleofector Technology is amenable to co-transfections, which opens the door to multiple cutting-edge applications. “It has been proven to work well for non-viral iPSC generation from somatic cells (e.g., fibroblasts, PBMCs, or CD34 cells) via co-transfection of three to five episomal vectors encoding the reprogramming factors,” says Toell. Scaling up transfection with the 4D-Nucleofector System, Lonza’s technology is used to support high-throughput or larger-volume co-transfections for applications such as CAR-T cell generation, and genome editing using ZFN, TALEN, and CRISPR. “For CAR-T cell generation and genome editing, researchers now try to transfect the modifying component (i.e., the nuclease or transposase) as mRNA or even protein, in order to minimize unwanted off-target effects,” she says.

Viral methods

Viral transfection, or transduction, is an efficient way to introduce genes into cells using vectors such as adenoviruses or retroviruses. Viral transduction is commonly used in cell lines, primary cells, immune cells, in vivo applications, and in cell types not amenable to other transfection methods. “Slowly dividing or quiescent cells, some immune cell types, and cells generally known as hard-to-transfect such as primary human T cells, may be more efficiently transfected with electroporation or viral transduction,” explains Tseng.

Indeed, choosing any transfection method is a balancing act. “For example, retroviruses are the most commonly used and accepted biological method for transducing immune cells (e.g., T cells), but have safety implications, both from a research-only and a clinical perspective,” says Lindy O’Clair, Head of Incucyte^® Product Management at Sartorius. “Other methods such as electroporation and chemical liposomes alleviate safety and immunogenicity concerns, but they risk cell viability, which is critical to maintain as we move into more relevant, primary cell models.”

Even though no method is problem-free, the potential pitfalls of most common transfection methods are well-understood today, so doing your homework can prevent unpleasant surprises. “No single system is perfect, so until a transfection method emerges that can balance efficiency while minimizing cell perturbation, scientists will continue to evaluate priorities and select an approach that is reproducible for their application needs,” says O’Clair, including consideration of cell type, insert size, and potential drawbacks of each method.

Look for future advances in transfection methods as researchers attempt to use gene editing in primary cells to treat genetic diseases. “The scientific community is advancing their understanding of how genes function and how to modify these genes to treat genetic disorders, but they are still limited by the tools used to manipulate cells,” says O’Clair. “I want to highlight the need for technologies to address the transition to more biologically-relevant primary cells and the utilization of gene editing.”