Transfection has evolved into one of cell biology’s foundational processes and, through the introduction of protein-coding genes into suitable expression systems, for the manufacture of biopharmaceuticals. The term “transfection” originally described gene transfer through viral infection, a process now known as transduction. Today transfection describes the non-viral transfer of genes, including siRNA, miRNA, large RNA, and oligonucleotides.

The most common transfection methods involve cationic lipids, polymers, or liposomes that encapsulate negatively charged nucleic acids and deliver them to cells. Transfection reagents neutralize electrostatic repulsions between genes and cell membranes, and help the reagent-nucleic acid complex to overcome the cell membrane’s physical barrier. Endocytosis often depends on recognition of the transfection complex, or reagent-nucleic acid construct, by cell surface receptors.

Multi-step process

Optimizing transfection requires close examination of the entire process, from selection of transfection method to protein expression. Intervention or improvement occurs at any step along the process, for example:

  • Cell type, nucleic acid, and protein/trait of interest
  • Choice of mechanical or chemical transfection
  • Transport of nucleic acid from endosome to functional locations in the cell
  • Nuclear translocation of DNA
  • Effect of transfection and/or protein expression on cell health and function

Because most of these factors are under the control of target cells themselves and are therefore poorly controlled or understood, developers of transfection protocols focus primarily on getting transfection complexes into cells. Thus the emergence of mechanical methods as alternatives to chemical transfection. These methods include electroporation, microinjection, biolistic delivery via gene guns, or magnetic-assisted transfection depending on the cell type and application.

Reagent-based chemical transfection, available through commercial reagent kits, dominate transfection due to the method’s simplicity and applicability to a wide range of cell types.

“The composition and physical characteristics of transfection complexes are critical for successful gene transfer,” says Miguel Dominguez, global distribution manager at Mirus Bio. The increasingly common use of non-traditional cell lines, including primary cells and stem cells, which may not readily incorporate transfection complexes, underscores the importance of transfection optimization.”

Difficult-to-transfect cells

Mechanical transfection has emerged in large part to accommodate difficult-to-transfect cells. For example OZ Biosciences, whose flagship product is its proprietary Magnetofection™ platform, specializes in stem cells and primary cells such as neurons, endothelial, and microglial cells.

Many factors contribute to transfection difficulty or success, including cellular binding and internalization of reagent-gene complexes, release of nucleic acids into the cytoplasm, the gene’s nuclear uptake and expression, plus the cell’s health, metabolic activity, and division rate. “The primary mechanism for genes entering cells involves cell surface receptors that latch onto the cell-like liposomal reagents, triggering receptor-complex interactions and internalization,” says Romuald Arnaud, business manager at OZ Biosciences. “Easily transfected cells are covered with these receptors and have other desirable features such as rapid division, high endocytosis, and high metabolic activity.”

Immature cells, including stem cells and uncommitted progenitor cells, lack these characteristics. Similarly primary cells—increasingly employed as models in drug discovery, toxicology, and basic research—do not divide, have a lower internalization capacity, and often lack the ability to bind to transfection complexes.

Transfection difficulty is not limited to immature or primary cells. HeLa, one of biology’s workhorse cell lines, is refractory to high transfection efficiency using chemical methods. Cell lines that have been cultured too long or transfected multiple times might resist transfection as well. “A cell culture’s age, confluence, and passage number are additional factors that render them difficult to transfect,” Arnaud adds.

Magnetofection, a mechanical transfection method that enables cells to incorporate transfection complexes without physically damaging the cells, uses typical metallic nanoparticles coated with cationic molecules that complex with nucleic acids, including naked, packed, or virus-enveloped moieties, by electrostatic and hydrophobic interactions. Metal-bound complexes associate loosely with cells, but through the influence of a magnet placed beneath the culture dish, are drawn down or concentrated onto the cell surface, and eventually internalized.

In contrast to gene guns, electroporation, sonoporation, and other mechanical techniques Magnetofection does not compromise the cell membrane.

“This is not forced internalization, which while effective in getting genes into cells, results in a tremendous level of cell death,” Arnaud explains. “Magnetofection allows cells to internalize transfection complexes with the least amount of stress, delivering high levels of nucleic acid, which leads to a higher chance of obtaining transgene expression.”

Another advantage to Magnetofection and of mechanical methods generally—is consistency. Once a transfection is optimized for gene incorporation, protein yield, or any other desired trait, conditions are easily repeated to give identical results.

Advantages of low-volume prep

In addition to benefits of higher protein yield and expression, optimizing transfection can reduce the quantity of expensive DNA and reagents consumed, and sometimes increase a method’s scope to previously inaccessible cells.

A method developed by Promega, using its ViaFect™ transfection reagents, forms the DNA/reagent complexes in lower volumes compared with standard protocols. According to the company, the low-volume approach offers improved transfection efficiency in some cell lines, including HEK293, and reduces reagent consumption while maintaining cell health.

Brad Hook, manager for scientific applications at Promega, explains the benefits of low-volume preparation: “We showed improved efficiency in forming the DNA/reagent complex by using smaller-than-standard volumes. Instead of diluting the DNA nearly 1:100 before adding the reagent, we diluted 1:25, added reagent, incubated, then diluted the formed complex. This approach significantly improved transfection efficiency in some cell lines, including HEK293. For other lines, low-volume complex formation provided an option to reduce the amount of transfection reagent or DNA used while maintaining similar transfection efficiency.”

Hook notes that while transfection reagent vendors supply information on optimal transfection conditions for a particular cell line, such data should only serve as a rough guide.

“Optimization is typically required to arrive at the best transfection conditions for cells growing in a particular lab. Things like passage number and overall cell health, which affect transfection efficiency, vary from lab to lab. Assessing cell health as part of the optimization process is critical since toxicity caused by the transfection process can significantly affect whatever downstream biology you’re measuring. Conditions that provide the highest efficiency while keeping the cell population healthy are ideal.”