Primary Cells Pose No Problem with These Transfection Tools

Modern molecular biology relies heavily on the ability to introduce genetic material into cells. But this can be a challenge, especially for non- or slowly dividing cells, such as primary cells.

There’s no shortage of tools to help, but if you’re working with primary cells, you likely have some optimization ahead of you. Here we look at selected reagents and equipment to get you started.

Transient transfection

Researchers have three routes for delivering nucleic acids to cells. The first comprises chemical methods, which make negatively charged nucleic acids less repulsive to the negatively charged cell and facilitate uptake by phagocytosis, pinocytosis or fusion of the coated particle with the cellular membrane. The second route includes physical methods such as  electroporation and biolistic, magnetic and more esoteric methods that effectively poke holes in the cell and/or physically drive nucleic acid inside. The final approach, delivering the nucleic acid using a virus (or virus-like particle), often called transduction, is often a sure-fire way of getting DNA into and expressed by the cell but typically requires more expertise, time and biosafety precaution, and researchers doing translational work often shy away from it.

For many researchers, the goal often is a stable cell line expressing a gene or transcript of interest. But even when all goes according to plan, establishing a stable cell line can be a time-consuming enterprise. The DNA containing your gene(s) of interest must make it into the nucleus, integrate into the host genome (or, in the case of an episome, replicate on its own) and then get transcribed and translated.

Given all those steps, it can take six to eight weeks before you’re ready to try antibiotic selection, notes Dmitriy Ovcharenko, CEO of Altogen Labs, which offers pre-clinical research services, including generation of cell lines. Researchers therefore will typically test their plasmid constructs in a transient transfection first. “You can expect protein expression in 48 to 72 hours, sometimes 96 hours post-transfection,” Ovcharenko says. This step also help researchers determine if the cell will have problems tolerating what the plasmid encodes, be it protein or even miRNA.

Delivering mRNA or siRNA (rather than DNA plasmids) can provide a quick taste of what a particular transcript does as well, especially for nondividing or otherwise difficult-to-transfect cells like neurons and primary T cells. Although the effect will only last for perhaps a few days, RNA is generally easier to transfect, because it doesn’t need to enter the nucleus to yield results.

Stable transfection

For long-term expression, you need a transgene that sticks around and replicates along with the host. The standard method of achieving that goal in primary cells has been electroporation. And although this remains the fallback procedure, companies offer myriad reagents that can and have been used to stably transfect even difficult-to-transfect cells.

Some of these are tried-and-true multipurpose reagents that have been on the market for some time (or updated versions of these), such as Promega’s FuGENE® and Life Technology’s Lipofectamine® reagents. Others (such as Incella’s ScreenFect®A and ATCC’s TransfeX™) represent newer launches.

Many vendors also have formulated reagents and protocols specifically for given cell types. For example, EZ Biosystems’ web site lists some 33 distinct Avalanche™ transfection-reagent products for primary tissue, from human artery endothelial cells to urethra epithelium and mouse embryonic fibroblasts. Similarly, Cell Applications offers such kits as the Cytofect™ Fibroblast Transfection Kit with optimized protocols for primary dermal, cardiac and lung fibroblasts.

There are dozens of transfection reagents and kits on the market, typically consisting of proprietary lipid-based and/or polymer-based formulations, often combining several reagents. “Cell-specific reagents or reagents directed to primary cells contain specific formulations that have been optimized and tested for those types of cells, to provide … optimal transfection efficiency while maintaining cell viability,” notes Teresa Rubio, R&D manager of the Cell Biology Business Unit at Bio-Rad Laboratories.

Unfortunately, while product literature inevitably boasts high efficiency and low toxicity, there is no easy way to gauge which reagents will work with which cells. Labs inevitably need more than one option, however, as it simply isn't possible to have one reagent for all applications. “In reality, we do not have a complete enough understanding of the biochemical mechanisms of cellular transfection to allow us to predict a priori which molecule(s) will best facilitate transfection of a given cell type, for a given application,” says Pavel Levkin, Incella’s chief scientific officer. “That is why it is important to optimize transfection for different cell types and reagents if you want to have the highest efficiency.”

Incella is endeavoring to understand the structure/function relationships that make a difference in delivering nucleic acid into the cells, Levkin says. What, for instance, are the reagent and cellular properties, such as nanoparticle size or the presence of serum, that distinguish good and bad transfection conditions for a given cell type? The company has developed and screened hundreds of cationic lipid-like molecules to find optimized conditions for different cell types and plans to roll out dedicated transfection products later this year “to cover all different niches that scientists are interested in or working on.”

Step by stepwise

The first thing Ovcharenko recommends when a researcher has primary cells to transfect is to identify a set of pre-optimized transfection reagents—those claiming a broad cellular range as well as those specifically optimized for that particular cell type, if any. And then, simultaneously compare as many as possible.

“If that does not provide high enough transfection efficiency,” he says, “the second step is to try electroporation—assuming there is capability of the lab to do that.”

Like chemical reagents, several electroporators are on the market, and various kits and reagents have been optimized for different cell types. Some systems, like Lonza’s Nucleofector™, use proprietary pre-set programs. Most others are open systems that allow users to program electrical parameters, such as wave form and pulse length.

“In terms of what they do to the cell, they’re very similar—you’re actually electrocuting your cells,” notes James Lovgren, associate director of product management at Thermo Fisher (which recently acquired Life Technologies), who is responsible for transfection products. The trick is getting those parameters right, “balancing delivering the nucleic acid in an efficient way while at the same time being gentle on the cells, so that they survive the process.”

For Randy Cron, professor of pediatrics and medicine at the University of Alabama at Birmingham, the instrument of choice for transfecting primary T cells is the Nucleofector. The system punches holes in both the cytoplasmic and nuclear membranes, he explains. This is an advantage for getting nucleic acid into the nucleus, but it also leads to calcium flux and an increased expression of CD4+ T cell activation markers [1]. Cron cautions users to take this into account when planning and interpreting their experiments.

Ovcharenko says that if electroporation doesn’t provide high transfection efficiency, “probably the last resort is a viral method”—with the caveat that though viral delivery has the potential to provide the highest efficiency, it also tends to require a lot of cloning and optimization as well as safety precautions that are not always necessary for other methods.

Getting transgenes to stick around and be expressed in primary cells can be a tricky endeavor, but don’t get discouraged. A lot of the legwork has already been done by your fellow researchers—make sure to check the literature—as well as by vendors both large and small. Reagents, instrumentation and protocols are available for many cell types, and these often can be adapted to suit novel situations.

Reference

[1] Zhang, M, et al., “The impact of Nucleofection® on the activation state of primary human CD4 T cells,” Journal of Immunological Methods, 408:123–31, 2014. [PubMed ID: 24910411]