Nucleic acid direct delivery to fibroblasts: a review of nucleofection and applications.

The fibroblast is one of the ideal target cell candidates for cell-based gene therapy approaches to promote tissue repair. Gene delivery to fibroblasts by viral transfection has been confirmed to have high transfection efficiency. However, in addition to immunogenic effects of viruses, the random integration of viral genes may damage the genome, affect the cell phenotype or even cause cancerous mutations in the transfected cells. Due to these potential biohazards and unknown long-term risks, the clinical use of viral transfection has been very limited. In contrast, initial non-viral transfection methods have been simple and safe to implement, with low immunogenicity, insertional mutagenesis, and risk of carcinogenesis, but their transfection efficiency has been relatively low. Nucleofection, a more recent non-viral transfection method, now combines the advantages of high transfection efficiency and direct nucleic acid delivery to the nucleus with a high safety.Here, we reviewed recent articles on fibroblast nucleofection, summarized different research points, improved methods and application scopes, and opened up ideas for promoting the further improvement and development of fibroblast nucleofection to meet the needs of a variety of disease research and clinical applications.

High Efficiency Low Cost Fibroblast Nucleofection for GMP Compatible Cell-based Gene Therapy.

Background: Dermal fibroblast is a powerful tool for the study of ex vivo DNA delivery in development of both cell therapy and tissue engineering products. Using genetic modification, fibroblasts can be diversely adapted and made suitable for clinical gene therapy. In this study, we first compared several non-viral transfection methods including nucleofection in rat and human primary dermal fibroblast. In addition, the original protocol for nucleofection of primary mammalian fibroblasts was modified in order to achieve the highest possible transfection efficiency, as determined by flow cytometry analysis of the green fluorescent protein (GFP) expression. Results: the results showed that transfection performance of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) yielded the best transfection efficiency with rat dermal fibroblasts and ITS (insulin, transferrin, and sodium selenite solution) was comparable to the standard nucleofection solution for human dermal fibroblasts. Conclusion: Our results suggest a promising application of the modified nucleofection method for GMP compatible therapeutic translational medical research.

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.

BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.