This study aimed to develop a suitable plasmid transfection protocol for human cardiac fibroblasts (HCFs). After expanding the HCFs to passage 8–9, the researchers tested 12 commercially available chemical transfection reagents (R2 to R13). Transfection efficiency was assessed by GFP fluorescence, showing that R3 (17.65%) and R4 (21.7%) had the highest efficiencies, while others had less than 5%. Cell viability was also evaluated using propidium iodide (PI) staining, with R7 (14.05%) and R12 (13.53%) showing the lowest cell death, compared to R3 (23.47%). Based on these results, R7 and R12 were selected for further optimization, with R4 kept as a potential candidate despite lower cell viability due to its high transfection efficiency.
Effect of plasmid purification methods on fibroblast viability
The impact of different plasmid purification methods (M1-M8) on human cardiac fibroblast (HCF) viability after transfection with R4-mediated plasmids. Results showed that plasmids purified with methods M1-M8 led to a ~50% reduction in cell viability compared to non-transfected controls, with M7 showing slightly better survival rates. LDH release indicated greater cytotoxicity from M1, M2, and M8 compared to other methods. Gel electrophoresis showed that M7 and M8 had lower yields of desired products compared to the centrifugation-based M6 method. Transfecting cells with plasmid alone did not affect viability, but R4-plasmid complexes significantly decreased survival, regardless of transgene expression. Increasing plasmid concentration led to a proportional decrease in viability. Adding E. coli RNA or LPS did not cause cytotoxicity, except at high concentrations. DNase I treatment improved survival rates, suggesting that nucleic acids rather than impurities were more detrimental to viability. The study also identified an upregulation of inflammatory cytokines and DNA sensors (CGAS and STING) as a result of transfection. Based on these findings, M5 was selected for larger-scale optimizations, and M3 for small-scale experiments.
This study systematically evaluated 100 conditions for R4, 45 for R12, and 9 for R7 to optimize the plasmid transfection protocol. Key findings included that the timing of media changes significantly affected gene delivery. For R4, the highest luciferase activity occurred when changing the media 4 hours post-transfection. For R12, increasing R7 volume and optimal media change timing (4-8 hours) improved luminescence. R7 showed peak luminescence with 100 ng plasmid and 0.30 μl reagent at 8 hours. A trade-off between transfection efficiency and cell viability was observed, with a negative correlation between luciferase activity and cell survival. The P26 combination was chosen as the optimal condition, providing the best balance of viability and gene delivery, with sustained high expression for 24-72 hours.
Promoter assay using human primary cardiac fibroblasts
Discussion
The study presents the first optimized plasmid-based gene introduction protocol for primary human cardiac fibroblasts (HCF), offering an alternative to viral vector systems. While viral vectors typically provide high expression levels, they require complex production and may introduce confounding effects from viral sequences. The optimized plasmid method, using purified plasmids from tube-scale E. coli cultures, demonstrated similar cell viability and robust gene expression, with luciferase assays showing significant upregulation (100–350 times background). The method was versatile, enabling sustained gene expression for up to 72 hours, making it ideal for medium-term reporter assays and signaling pathway investigations.
Key protocol parameters include maintaining a seeding density of 1.82 x 10^4 cells/cm², which ensures optimal proliferation and reproducibility during experiments. The study also optimized timing for media changes and reagent use, with the Viafect (R12) reagent achieving maximum luciferase activity from 48–96 hours post-seeding, and Lipofectamine 3000 (R7) showing peak activity at 48 hours post-transfection.
Furthermore, the study addressed potential challenges in plasmid introduction, such as inflammatory cytokine induction and apoptosis from overexpressed foreign proteins, E. coli-derived components, or plasmid-related factors, providing insights into minimizing cell toxicity. The methodology is applicable to experiments investigating fibroblast function, gene regulation, and cardiac disease.
URL Link:
Novel and effective plasmid transfection protocols for functional analysis of genetic elements in human cardiac fibroblasts | PLOS ONE