Fibroblast cells from humans and other mammals are often used in biological research utilizing cell transfection methods. Some of the commonly used fibroblast cell lines are 3T3, a murine fibroblast line established in 1962 at the NYU School of Medicine. 3T3 cells were originally sourced from Swiss albino mice embryos and spontaneously immortalized. Another commonly used cell line is MRC-5, derived from human lung tissue.
Fibroblast Transfection
Transfection is the introduction of foreign molecules and genetic material into cultured cells. Transfections can be transient or stable, and the process is utilized in biological research to study gene function and to model the effect of gene therapy on cellular expression. In transient transfections, the short term impact of altered gene function is examined, with high expression persisting for 72 hours or less. Stable transfection requires that target genes be incorporated into the genome of a cell culture in such a way that expression continues throughout the life of the cell line and during each round of cell selection during subculturing.
pDNA and mRNA can be introduced via transfection, as well as siRNA and miRNA, which disrupt the development of gene products. This method of post-transcriptional gene silencing has a huge potential in terms of drug therapies that target genetic disease at the source, targeting the gene itself with a high degree of specificity, rather than relying on symptomatic or higher level treatment.
There are a great variety of carrier molecules on the market today which enable scientists to introduce genetic material into cancer or primary cells through non-viral means. This can be accomplished using immortalized cell culture lines, but also in vivo. However, all cells are different, and require different transfection reagents, carrier molecules, transfection protocols and reagents in order to successfully express a vector gene of interest. For links to resources for transfection and biological services, products and reagents, see below.
Fibroblast Transfection Protocol
A pre-optimized Fibroblast Transfection Kit is available from Altogen Biosystems, which includes:
- Fibroblast Transfection Reagent (0.5 ml / 1.5 ml / 8.0 ml)
- Transfection Enhancer (0.5 ml), and
- Complex Condenser (0.5 ml)
The kit is optimized for transfection of DNA and RNA into Fibroblast cells following either a standard or reverse transfection. The optimized protocol for a 24-well plate to transfect Fibroblast cells is described below:
- Plate 7,500-12,000 Fibroblast cells per well in 0.5 ml of complete growth medium 12-24 hours prior to transfection
- Wash with 1xPBS and add 0.5 ml of fresh growth medium
- Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and (referred to a final volume including growth medium)
- 750 ng DNA (or mRNA), or
- 30 nM – 50 nM of siRNA (or microRNA)
- Incubate transfection complexes at RT for 15-30 minutes
- Optional: Add 2 µl of Complex Condenser. This reagent increases transfection efficiency by reducing the size of transfection complex; however, it may increase cell toxicity
- Add prepared transfection complexes to 0.5 ml of complete growth medium with Fibroblast cells (from step 2)
- Incubate cells at 37ºC in a humidified CO2incubator
- Assay for phenotype or target gene expression 48-72 hours after transfection
Optional: Adding Transfection Enhancer reagent can increase the transfection efficiency. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection
If the viability of Fibroblast cells being transfected is affected at 16-24 hours post-transfection, changing the growth medium and eliminating redundant exposure of cells to transfectant can decrease the level of cytotoxicity
Links:
Fibroblast Transfection Kits – Purchase Fibroblast Transfection Reagent
Protocols, techniques, methods, in vivo transfection – siRNA Transfection
Xenograft.net – Preclinical biology CRO research services
Cell Transfection Resource
Transfection. Cells and Molecular Biology Research Methods, Protocols, and Lab Techniques
Research Articles:
- Similarities between fibroblasts and embryonic stem cells: This study compares MRC-5 fibroblast cells to embryonic stem cells in terms of morphology, growth rate and gene expression. In addition, the researchers develop a serum-free culture system that maintains the MRC-5 cells in a proliferative state similar to that of the embryonic cells. [LINK]
- Response of fibroblast cell lines to hydrogen peroxide: Researchers study the effect of hydrogen peroxide on human fibroblast cells, as peroxide has been previously shown to induce apoptosis or senescence in cultured cells. Results show that hydrogen peroxide in increasing concentrations produce a transient shock state, then senescence or apoptosis.
- Efficiency of different transfection methods in mouse embryonic fibroblast (MEF) cells: Although viral gene-delivery methods are highly efficient, MEF cells are difficult to transfect, and challenges have led to the improvement and use of non-viral gene-delivery methods. This study compares the effectiveness of different gene-delivery methods. Results show that two gene delivery methods, electroporation and magnetofection (when combined with a lipid reagent), are the most efficient in MEF cells. [LINK]
Fibroblast Cell │ Culture Protocol │ Fibroblast Transfection Information