An Overview of Cell Transfection Methods
This article presents the state-of-the art of transfection methods from their working principles to a detailed comparison of each technique with its pros and cons. With this overview, we will introduce the future of transfection methods: direct intra-nuclear injection using the FluidFM technology that enables vector-free single-cell transfection.
Go straight to: Definition | Types of transfections | Methods of transfection | How to choose the right transfection method
What is the principle of transfection in biology?
In brief, cell transfection regroups the general process of introducing DNA, RNA or proteins inside cultured cells.
Transfection is a widespread cell culture technique and an effective tool to analyze gene functions and gene products in cells. Transfection describes the mechanism in which external nucleic acids, peptides or proteins are delivered into a eukaryotic cell to study or control biological processes. [1,2] The success of a transfection depends on the method, delivery efficiency of the material into cells, and cell’s capacity to integrate the molecules. The delivery efficiency is dependent upon the genetic material quantity and quality, the incubation time, and the ratio of transfection reagent to DNA. Parameters such as the origin, type and the passage of transfected cells, and the presence or absence of serum in the cell culture are also key to make a successful delivery. [3]
What are the two types of transfections?
Stable & Transient transfections
Transient transfection does not require integrating nucleic acids into the host cell genome. [6,7] The latter is commonly used for short term expression of a desired gene that lasts a few days. Good examples of applications include research of gene expression, gene silencing studies and analysis of recombinant proteins.
Stable transfection refers to sustaining long-term expression of a transgene by integrating foreign DNA into the host nuclear genome or maintaining an episomal vector in the host nucleus as an extra-chromosomal element [6,7]. The integration of the forgein DNA is randomly localized within the host genome which can cause unwanted genetic modification among others. Alternatively, CRISPR gene engineering enables safer DNA integration by targeting specific regions of the genome.
Chemical & Physical Transfections
Chemical transfections comprise a number of steps from the plating of the cells at sub-confluency, preparing the transfection reagent / DNA complex immediately before transfection, to the assessment of the construct expression. Oppositely, through direct transfer, which involves breaching the membrane to distribute the material, DNA and RNA are transported across the membrane in physical transfection procedures.
Applications of Cell Transfection
Biological research
Gene and cell therapy
Bioprocessing
Agriculture & Plant Biotechnology
What are the different methods of transfection?
Lipofection, Electroporation, Viral transduction, Calcium Phosphate Co-precipitation, DEAE-Dextran Transfection, Micro-Injection...
Lipofection - a standard chemical transfection - uses liposomes, small molecules that can fuse with cell membranes and are able to release their contents. Common applications comprise research in Oncology notably to target specific genes (oncogenes) via small molecule-based drugs or gene silencing using RNAi technology.
Electroporation refers to a mechanical transfection where an electrical pulse is applied through an electroporation cuvette to create temporary pores in the cell membrane. The electrical pulse applied and the effectiveness of the technique itself depends on DNA concentration and the type of cells studied.
Viral transduction also known as virus-mediated transfection describes the use of viral vectors to transfer nucleic acids into the cells. [8-9]
Calcium Phosphate Co-precipitation describes the process of mixing DNA with calcium chloride in a buffered saline/phosphate solution to generate a calcium-phosphate–DNA co-precipitate, which is then dispersed onto cultured cells. Calcium phosphate facilitates the binding of the condensed DNA in the co-precipitate to the cell surface, and the DNA enters the cell by endocytosis. The process efficiency is limited by a number of parameters including small changes in pH, temperature, and buffer salt concentrations. [10]
DEAE-Dextran Transfection employs the diethylaminoethyl-dextran (DEAE-dextran) - a polycationic derivative of dextran (a carbohydrate polymer) to facilitate transfection. When mixed with the DNA, the resulting complex is carried into contact with the negatively charged plasma membrane by the excessive positive charge provided by the polymer.
Microinjection is commonly used to introduce DNA and RNA into single cells. With the aid a micromanipulator and microscope, the DNA or RNA is directly inserted into the cytoplasm or nucleus. This is typically time consuming, but results in very high transfection efficiency. A sub-category of injection called "nanoinjection" is enabled with the FluidFM Technology.
Due to the gentleness of the technique, the term "direct intra-nuclear injection" was used, and the expression "single-cell biopsy" was developed to describe the use of the same approach to perform extraction of genetic information from the cell without damaging the cell. Electroporation and cell microinjection are two good examples of physical transfection methods that are harsh on cells due to disruption of the cellular membrane and often results in cell death. Those are therefore inappropriate options for cells that have been traditionally difficult to transfect, unlike the method of the direct intra-nuclear injection.
Comparison of Cell Transfection Methods
To address those challenges, Cytosurge employed its proprietary patented technology - the FluidFM technology - to offer a unique tool to perform direct intra-nuclear injection - the FluidFM OMNIUM.
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Lipofection | Electroporation | Viral Transduction | Microinjection | FluidFM Nanoinjection |
| Hard-to-transfect cells |
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| Multiplex Editing |
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| HDR - Large knock-in |
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| Genetic screens |
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| Autologous gene / cell therapy |
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| Allogeneic cell therapy (off-the-shelf) |
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| Transfection of millions of standard cells |
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| Generation of animal models |
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Table 1: Comparison of traditional transfection methods versus the FluidFM Nanoinjection - direct intra-nuclear delivery method.
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Not recommended |
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Works but with drawbacks |
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Works but not optimal in every situation |
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Method of choice |
Knowledge Center
References
[1] Chong, Zhi Xiong, Swee Keong Yeap, and Wan Yong Ho. "Transfection types, methods and strategies: A technical review." PeerJ 9 (2021): e11165.
[2] Kim & Eberwine (2010) Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. 2010;397(8):3173–3178. doi: 10.1007/s00216-010-3821-6.
[3] Fus-Kujawa, Agnieszka, et al. "An overview of methods and tools for transfection of eukaryotic cells in vitro." Frontiers in Bioengineering and Biotechnology (2021): 634.
[4] Kim & Eberwine (2010) Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. 2010;397(8):3173–3178. doi: 10.1007/s00216-010-3821-6.
[5] Stepanenko & Heng (2017) Stepanenko AA, Heng HH. Transient and stable vector transfection: pitfalls, off-target effects, artifacts. Mutation Research/Reviews in Mutation Research. 2017;773:91–103. doi: 10.1016/j.mrrev.2017.05.002.
[6] Lufino, Edser & Wade-Martins (2008) Lufino MMP, Edser PAH, Wade-Martins R. Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression. Molecular Therapy. 2008;16(9):1525–1538. doi: 10.1038/mt.2008.156.
[7] Riedl et al. (2018) Riedl S, Kaiser P, Raup A, Synatschke C, Jérôme V, Freitag R. Non-viral transfection of human T lymphocytes. Processes. 2018;6(188):1–17. doi: 10.3390/pr6100188.
[8] Glover, Dominic J., Hans J. Lipps, and David A. Jans. "Towards safe, non-viral therapeutic gene expression in humans." Nature Reviews Genetics 6.4 (2005): 299-310.
[9] Pfeifer, Alexander, and Inder M. Verma. "Gene therapy: promises and problems." Annual review of genomics and human genetics 2.1 (2001): 177-211.
[10] Graham, Frank L., and Alex J. Van Der Eb. "A new technique for the assay of infectivity of human adenovirus 5 DNA." virology 52.2 (1973): 456-467.