James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
Transfection is one of those do-or-die processes in mammalian cell research: Without it, many experimental findings might never have been made.
Researchers have numerous options for DNA delivery, including viral delivery, electroporation, liposomal transfection, and more. Choosing the best one depends on the cell type, nucleic acid type (plasmid DNA or siRNA, for instance) and transfection efficiency needed for optimal results.
Stem cells, though, provide several unique challenges. Pluripotent stem cells, for instance, often are grown in colonies, leading to cell morphologies that decrease transfection efficiencies. When separated into single cells, they are more susceptible to toxicity. Other stem cell types are affected by their culture environments, and changes to growth rate or culture health can impact transfection efficiency. Plus, says Kerry Mahon, manager of scientific development at Stemgent, “Materials that have been used to transfect other cell types (such as common cationic lipids) lead to low efficiency or toxicity on stem cells.”
Still, there’s no shortage of reagents and protocols available for the stem cell community. Here, we review some of the more popular, non-viral options.
Neon
According to Xavier de Mollerat du Jeu, senior staff scientist for cell biology at Life Technologies, one of the key problems with transfecting certain types of stem cells (such as pluripotent cells) has to do with their tendency to form spheroids. “There are challenges with stem cells, mainly because of the way they grow (in clusters). So, transfection of the cells in the outer perimeter of the colony is efficient, but this is not the case for the cells in the center of the colony.”
The company offers several transfection tools to address a variety of cell types. Released in January, Lipofectamine® 3000 is Life Technologies’ newest lipid transfection reagent – the latest in a 20-plus-year-old product line. But a better option for primary and stem cells, according to company literature, is the Neon® Transfection System. The electroporation-based Neon is highly efficient and versatile, offering up to 90% efficiency in many cells including in stem cells.
Non-liposomal, chemical methods
Mirus Bio TransIT®-2020 and TransIT-LT1 transfection reagents uses polymers to deliver nucleic acid to hard-to-transfect cell types such as stem cells and primary cells in a highly efficient manner with low toxicity. Non-liposomal in nature, TransIT requires no medium changes and can even be carried out in the presence of serum.
Another popular option is Xfect™, a biodegradable polymer from Clontech that is recommended for most cell types, including stem cells. Xfect Transfection Reagent will transfect human adipose derived stem cells at 90% efficiency and human mesenchymal stem cells at 40% efficiency. Another formulation, called Xfect mESC Transfection Reagent, works particularly well with mouse embryonic stem cells. “The challenges for transfecting stem cells are obtaining high efficiencies and retaining the normal behavior of the cells,” says Baz Smith, senior product manager at Clontech, which produces Xfect.
“I think most people are looking for transfection products that work with all cell types, but there is no product that does that,” says Smith. He explains that successful plasmid transfection relies on nuclear membrane breakdown at mitosis. If the cell is not dividing, the plasmid will not enter the nucleus to express a transgene regardless of the transfection reagent used. For this reason, viral transduction often is required for efficient delivery to many primary cell types.
According to Smith, Xfect offers a simple, single-tube protocol that, like the TransIT reagents, do not require researchers to remove media serum first. The logic is simple, Smith says: serum is good for cells, and healthy cells tend to transfect well.
Charge-based methods
QIAGEN’s transfection technologies are based on cationic lipids, which form positively charged spheres to which negatively charged nucleic acids adhere. These charged complexes may enter cells either by membrane fusion or endocytosis.
“Successful transfection is influenced by many factors,” says Andrea Stutte-Rabe, associate global product manager at QIAGEN, including cell line health and viability, nucleic acid quality, transfection reagent used, duration of transfection, and the presence or absence of serum. “QIAGEN´s transfection reagents are developed for highly efficient transfection with extremely low cytotoxicity,” he says.
According to Stutte-Rabe, QIAGEN transfection reagents, and particularly its Effectene and HiPerFect reagents, have been successfully applied to stem cells. “However, it is always a challenge to transfect stem cells,” he says, “therefore optimization steps need to be taken into account.”
Transfection efficiency is highly dependent on the overall health and growth rate of the cells. Therefore, when optimizing transfection – whether of stem cells or any other kind of cell -- all aspects of the culture environment need to be taken into consideration. That includes everything from such stem cell-specific variables as colony size, morphology and packing density, to the transfection conditions themselves –parameters like amount of nucleic acid, ratio of nucleic acid to transfection reagent, incubation time and concentration.
If you’re feeling overwhelmed, contact your vendor’s technical support line -- most transfection reagent suppliers can provide protocols for specific cell types, or at least point you in the right direction.