What Is Transfection? | 3D Cell Transfection | Corning

Transfection is the introduction of foreign DNA or RNA (nucleic acids) into cells. This procedure creates genetically modified cells that can be vital to biological research.

When considering the question, "What is transfection?", it's important to start with the basics. The DNA or RNA inserted into cells during transfection can exist in either a stable or transient form. Genes that are stably transfected persist within the cell. They integrate into and replicate with the host's genome. However, transiently transfected genes do not merge with the host's genome. As such, they are expressed for a limited time.

Scientists use this method to study gene products and how genes function in cells. It can inhibit or augment the expression of specific genes in cells, or it can be used to produce recombinant proteins.

Investigators can use transfection in a variety of ways in the lab. For example, transfected cells can be used for pluripotent stem cell generation, small interference RNA knock-down procedures, or gene therapy delivery to alleviate symptoms or cure disease.

According to a study in Analytical and Bioanalytical Chemistry, transfection can be achieved via three methods:

1. Biological: This technique uses genetically engineered viruses to introduce DNA or RNA into cells. The main biological method is viral transfection, also called transduction. Scientists frequently used viral transfection with cells that do not yield easily to other transfection methods.
2. Chemical: This strategy is used in contemporary research, and it relies on carrier molecules to either neutralize or convey a positive charge onto the DNA or RNA cells. These positively charged nucleic acids are attracted to negatively charged cell membranes. Examples of chemical transfection include calcium phosphate precipitation, cationic lipid transfection, cationic polymer transfection, and DEAE-dextran transfection.
3. Physical: This method delivers DNA or RNA cells directly to the cell's cytoplasm or nucleus without chemical carrier molecules. Examples of physical transfection include biolistic particle delivery, direct microinjection, electroporation, and laser-mediated transfection.

Unlike transfection which avoids viral infection to introduce DNA or RNA to a cell, transduction actively uses a viral vector to accomplish the same goal.

As a strategy, transfection and transduction can both generate stable or transient DNA expression. Alternatively, with RNA, transfection and transduction are always transient. Transfection works with adherent immortalized cells.

Advancing the uses of and knowledge around transfection has been a slow process. However, recent research has opened doors to greater transfection implementation.

In 2024, investigators explored ways to simultaneously support transfection efficiency and cell viability to leverage transfected stem cells and T cells with personalized cell therapy and immunotherapy. To accomplish this goal, they created an acoustothermal transfection method that applies acoustic and thermal effects to cells, making the cell membrane and nuclear envelope more permeable. This method delivers two large plasmid types into the nuclei of mesenchymal stem cells, enabling the regulation and expression of multiple complex genes.

There have also been other challenges around transfection and cell cultures. Most nonviral gene delivery studies have focused on identifying gene transfer mechanisms in 2D cell cultures. As a result, little is known about gene transfer in a 3D cell culture to date. However, some studies have shown that balancing cell migration with rate-of-matrix degradation can enhance gene transfer in 3D cultures and that cell-matrix interactions can be manipulated to modulate gene transfer.

That said, in recent years, researchers have invested in developing multiple 3D cell culture methods that enhance transfection processes. In 2019, investigators maximized the use of condensed mRNA as a nonviral alternative to producing therapeutic cells from patients' bone marrow. They used microparticle-mediated delivery of complexed mRNA. This tactic enabled higher cell metabolic activity and higher transfection in culture conditions, including 3D culture.

In addition, scientists pushed to perfect long-term gene silencing with siRNA in 3D culture. They determined that siRNA prepared with traditional reduced-serum media was excluded at the Corning^® Matrigel^® Matrix boundary. However, siRNA formed and delivered with standard serum-containing culture medium could permeate matrigel, spheroids, and organoids.

Cell line health and viability, nucleic acid quality, transfection reagent, transfection duration, and serum affect successful transfection. Cells must be uncontaminated, grown in a fresh medium, and kept in appropriate incubation conditions. Check transfection protocols for serum requirements, too, because some require serum-free conditions.

Transfect cells at 40–80% confluency. Too few cells can cause poor growth due to limited cell-to-cell contact, while too many cells can lead to a resistance in uptaking foreign nucleic acid.

Most chemical transfection reagents also have optimal time windows between 5 and 30 minutes, depending on reagent. The optimal time depends on cell line, transfection reagent, and nucleic acid.

No. The interactions of every cell type's specific properties and every transfection reagent are complicated. Consequently, any efforts to predict transfection efficiency are wasted. However, some empirical values show various cell types, such as primary cells, quiescent cells, and suspension cells, are difficult to transfect.

Genetic material with higher purity will routinely deliver better transfection outcomes. Identifying contamination by lipopolysaccharides (endotoxins) that are introduced by bacteria during the manufacturing process is critical. The innate immune system can detect even trace amounts, significantly impeding the transfection process. It is recommended to use commercial kits to remove endotoxins from genetic material.

Plasmid cleaning based on miniprep protocols is not recommended because:

• Promotors have unique expression rates.
• Certain gene characteristics may heavily affect cell physiology or trigger cell death.
• The genetic material's dimensions and tertiary structure also impact transfection success.

Transfection is a fundamental part of gene therapy and regenerative medicine. Continued work with stem cells and 3D cultures is vital to further the progression of precision and personalized medicine.

Learn more about 3D cell culture with Corning Life Sciences.