What Is Transfection | 3D Cell Culture | Corning

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Transfection is a powerful tool in biological research. It enables the study of gene products and the function of genes within cells. Methods used to achieve transfection fall into one of three categories: biological, chemical, or physical, according to Analytical and Bioanalytical Chemistry.

Researchers use transfection to inhibit or enhance specific gene expression in cells as well as to produce recombinant proteins. Examples include induced pluripotent stem cell (iPS cell) generation, small interference RNA (siRNA) knock-down procedures, and delivery of gene therapy to alleviate the symptoms of or even cure a disease.

Transfection Basics

Essentially, transfection is the process by which foreign nucleic acids are inserted into cells, thus producing genetically modified cells. These nucleic acids — DNAs and RNAs — exist within the cells in either stable or transient form. Transiently transfected genes are not integrated into the cell's genome and are only expressed for a limited period of time. Conversely, stably transfected genes are just that — they're stable and persistent within the cell and become part of the genome, replicating along with the host cell's genome.

Gene Transfer Methods

Scientists have developed several gene transfer methods for various cell types and purposes. The virus-mediated method is most commonly used in clinical research, but it can cause immunogenicity and cytotoxicity.

Chemical transfer, widely used in contemporary research, typically uses cationic polymers, cationic lipids, calcium phosphate, and cationic amino acids. The basic concept is that positively charged chemicals make complexes of the chemical and the nucleic acids, which are attracted to the negatively charged cell membrane. We don't quite understand yet exactly how these complexes pass through the cell membrane.

Physical methods include biolistic particle delivery, electroporation, laser-based transfection, and direct microinjection. Using mRNA offers several benefits over using DNA, again depending on the goals and purpose of the transfection process.

Enhancing the Process

Most studies on nonviral gene delivery have focused on identifying gene transfer mechanisms in 2D cell culture. But according to Integrative Biology, little is understood about the intracellular mechanisms involved in gene transfer in a 3D cell culture. Some studies show that balancing cell migration with rate-of-matrix degradation enhances gene transfer in 3D cultures and that cell-matrix interactions can be manipulated to modulate gene transfer.

Various 3D cell culture methods have been developed and studied in attempts to enhance transfection processes. For example, a 2019 study published in Molecular Therapy: Nucleic Acids optimized the use of condensed mRNA as a nonviral alternative in producing therapeutic cells from patients' bone marrow. The researchers used microparticle-mediated delivery of complexed mRNA, which "enabled higher cell metabolic activity and higher transfection" in culture conditions, which included 3D culture, Molecular Therapy reported.

A 2018 study in Scientific Reports aimed to overcome the challenge of long-term target gene silencing with siRNA in 3D culture. The scientists found that siRNA prepared with traditional reduced-serum media were excluded at the matrigel boundary, but siRNA formed and delivered using a standard serum-containing culture medium were able to permeate matrigels, spheroids and organoids.

Transfection is a critical procedure for gene therapy and regenerative medicine applications. 3D cells will play an important role in its future, as the development of 3D cell culture methods are vital to the advancement of precision and personalized medicine.