Successful transfection is influenced by many factors—the choice of the transfection method, health and viability of the cell line, number of passages, degree of confluency, quality and quantity of the nucleic acid used, and the presence or absence of serum in the medium can all play a part in the outcome of your transfection experiment. While it is possible to optimize specific transfection conditions to achieve high transfection efficiencies, it is important to note that some cell death is inevitable regardless of the transfection method used.

Cell type

The choice of which cell type to use for a transfection experiment may seem obvious, but it is a critical factor that is often overlooked. Since each cell type is likely to respond differently to a given transfection reagent or method, choosing the appropriate cell type and proper experimental design are necessary to maximize results.

While established continuous cell lines are easier to work with in the laboratory, they may not be the best choice for modeling in vivo processes because of the multiple genetic changes that they have undergone. However, if the purpose of the transfection experiment is high-level production of recombinant proteins, it is not important that the cell line represents the in vivo situation as long as the cell line can express sufficient quantities of recombinant proteins with proper folding and post-translational modifications. For example, transient transfection of suspension-adapted Expi293F™cells grown in Expi293™ Expression Medium enables researchers to produce, starting from the vector of interest, greater than 1 g/L of correctly folded and glycosylated recombinant proteins.

Primary cultures, on the other hand, are often used because they more closely mimic natural tissues. However, they typically have a limited growth potential and life span, and are more difficult to maintain in culture. When using primary cultures, it is important to maintain a largely homogeneous population of cells (for example, neuronal cultures should be enriched for neurons and suppressed with regard to glial cells) and use the cells as soon as practical.

In addition, biological properties of the cell type must be taken into consideration when designing transfection experiments. For example, some promoters function differently in different cell types and some cell types are not well suited to particular transfection technologies.

Figure 6.1 Cell line-dependent differences in transfection efficiency. Lipofectamine® 2000 reagent and Lipofectamine® 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well plate format, using 0.5 µg plasmid/well and the recommended protocols for each reagent. GFP expression was analyzed 48 hours posttransfection. Each condition was tested in triplicate, and the data points show the mean transfection efficiency plus standard deviation.

Cell health and viability

The viability and general health of cells prior to transfection is known to be an important source of variability from one transfection to another. In general, cells should be at least 90% viable prior to transfection and have had sufficient time to recover from passaging. We strongly recommend subculturing cells at least 24 hours before transfection to ensure that they recover from the subculture procedure and are in optimum physiological condition for transfection.

Cell cultures with immortalized cell lines evolve over months and years in the laboratory, resulting in changes in cell behavior with regard to transfection. Excessive passaging is likely to detrimentally affect transfection efficiency as well as total transgene expression level from the cell population as a whole. In general, we recommend using cells that have undergone less than 30 passages after thawing of a stock culture. Thawing a fresh vial of frozen cells and establishing low-passage cultures for transfection experiments allow the recovery of transfection activity. For optimal reproducibility, aliquots of cells of a low passage number can be stored frozen and thawed as needed. Allow 3 or 4 passages after thawing a new vial of cells.

Since contamination can drastically alter transfection results, cell cultures and media should be routinely tested for biological contamination (see Biological Contamination), and contaminated cultures and media should never be used for transfection. If cells have been contaminated or their health is compromised in any way, they should be discarded and the culture re-seeded from uncontaminated frozen stocks.

Confluency

For optimal transfection results, follow a routine subculturing procedure and passage cultures once or twice a week at a dilution that allows them to become nearly confluent before the next passage. Do not allow the cells to remain confluent for more than 24 hours.

The optimal cell density for transfection varies for different cell types, applications, and transfection technology, and should be determined for every new cell line to be transfected. Maintaining a standard seeding protocol from experiment to experiment ensures that optimal confluency at the time of transfection is reliably achieved. With cationic lipid-mediated transfection, generally 70–90% confluency for adherent cells or 5 × 105 to 2 × 106 cells/mL for suspension cells at the time of transfection provides good results.

Make sure that the cells are not confluent or in stationary phase at the time of transfection, because actively dividing cells take up foreign nucleic acid better than quiescent cells. Too high of a cell density can cause contact inhibition, resulting in poor uptake of nucleic acids and/or decreased expression of the transfected gene. However, too few cells in culture may result in poor growth without cell-to-cell contact. In such cases, increasing the number of cells in culture improves the transfection efficiency. Similarly, actively dividing cell lines are more efficiently transduced with viral vectors. When transducing a non-dividing cell type with viral constructs, the MOI (i.e., multiplicity of infection) may need to be increased to achieve optimal transduction efficiency and increased expression levels for your recombinant protein.

Media

Different cells or cell types have very specific medium, serum, and supplement requirements, and choosing the most suitable medium for the cell type and transfection method plays a very important role in transfection experiments. Information for selecting the appropriate medium for a given cell type and transfection method is usually available in published literature, and may also be obtained from the source of the cells or cell banks. If there is no information available on the appropriate medium for your cell type, you must determine it empirically.

It is important to use fresh medium, especially if any of the components are unstable, because medium that is missing key components and necessary supplements may harm cell growth.

For cell culture media information, see Media recommendations for common cell lines. Some cell lines and primary cells may need special coating materials (e.g. poly-lysine, collagen, fibronectin etc.) to attach to the culture plates and get the optimal transfection results.

Serum

In general, the presence of serum in culture medium enhances transfection with DNA. However, when performing cationic lipid-mediated transfection, it is important to form DNA-lipid complexes in the absence of serum because some serum proteins interfere with complex formation. Note that the optimal amounts of cationic lipid reagent and DNA may change in the presence of serum; thus, transfection conditions should be optimized when using serum-containing transfection medium.

When transfecting cells with RNA, we recommend performing the transfection procedure in the absence of serum to avoid possible contamination with RNases. Most cells remain healthy for several hours in a serum-free medium.

The quality of serum can significantly affect cell growth and transfection result. Therefore, it is important to control for variability among different brands or even different lots of serum to obtain best results. After testing the serum on your cells, keep using the same serum to avoid variation in your result. All Life Technologies™ and Gibco® products, including sera, are tested for contamination and guaranteed for their quality, safety, consistency, and regulatory compliance.

Antibiotics

In general, antibiotics can be present in the medium for transient transfection. However, because cationic lipid reagents increase cell permeability, they may also increase the amount of antibiotics delivered into the cells, resulting in cytotoxicity and lower transfection efficiency. Therefore, we do not recommend adding antibiotics to the transfection medium. Avoiding antibiotics when plating cells for transfection also reduces the need for rinsing the cells before transfection.

For stable transfections, penicillin and streptomycin should not be used in selective medium, because these antibiotics are competitive inhibitors of the Geneticin® selective antibiotic. When creating stable cell lines, allow 48 to 72 hours after the transfection procedure for cells to express the resistance gene before adding the selective antibiotic.

If using serum-free medium, use lower amounts of antibiotics than you would in serum-containing medium to maintain the health of the cells.

Type of molecule transfected

Plasmid DNA is the most commonly used vector for transfection. The topology (linear or supercoiled) and the size of the plasmid DNA vector influence the efficiency of transfection. Transient transfection is most efficient with supercoiled plasmid DNA. In stable transfection, linear DNA results in lower DNA uptake by the cells relative to supercoiled DNA, but yields optimal integration of DNA into the host genome.

Although other macromolecules such as oligonucleotides, RNA, siRNA, and proteins can also be transfected into cells, conditions that work for plasmid DNA need to be optimized when using other macromolecules.

Transfection method

There are a number of strategies for introducing nucleic acids into cells that use various biological, chemical, and physical methods. However, not all of these methods can be applied to all types of cells and experimental applications, and there is a wide variation with respect to transfection efficiency, cell toxicity, effects on normal physiology, level of gene expression etc. The ideal approach should be selected depending your cell type and experimental needs, and should have high transfection efficiency, low cell toxicity, minimal effects on normal physiology, and be easy to use and reproducible. For an overview and comparison of various transfection methods, see Gene Delivery Technologies.