Guo Tony J F, Liang Wan Yi, Singhera Gurpreet K, Memar Vaghri Jasmine, Leung Janice M, Dorscheid Del R
Centre for Heart Lung Innovation, St. Paul's Hospital, Providence Healthcare Research Institute, University of British Columbia, 1081 Burrard St, Vancouver, BC, V6Z 1Y6, Canada.
Department of Medicine, University of British Columbia, 2775 Laurel St, Vancouver, BC, V5Z 1M9, Canada.
BMC Biotechnol. 2025 Jan 23;25(1):10. doi: 10.1186/s12896-025-00945-x.
Chemical transfection is a widely employed technique in airway epithelium research, enabling the study of gene expression changes and effects. Additionally, it has been explored for its potential application in delivering gene therapies. Here, we characterize the transfection efficiency of EX-EGFP-Lv105, an EGFP-expressing plasmid into three cell lines commonly used to model the airway epithelium (1HAEo-, 16HBE14o-, and NCI-H292).
We used six common and/or commercially available reagents with varying chemical compositions: Lipofectamine 3000 (L3000), FuGENE HD, ViaFect, jetOPTIMUS, EndoFectin, and calcium phosphate. Using L3000, 1HAEo- exhibited the highest transfection efficiency compared to 16HBE14o- and NCI-H292 (1HAEo-: 76.1 ± 3.2%, 16HBE14o-: 35.5 ± 1.2%, NCI-H292: 28.9 ± 2.23%). L3000 yielded the greatest transfection efficiency with the lowest impact on cellular viability, normalized to control, with a 11.3 ± 0.16% reduction in 1HAEo-, 16.3 ± 0.08% reduction in 16HBE14o-, and 17.5 ± 0.09% reduction in NCI-H292 at 48-hour post-transfection. However, jetOPTIMUS had a similar transfection efficiency in 1HAEo- (90.7 ± 4.2%, p = 0.94), but had significantly reduced cellular viability of 37.4 ± 0.11% (p < 0.0001) compared to L3000. In 16HBE14o-, jetOPTIMUS yielded a significantly higher transfection efficiency compared to L3000 (64.6 ± 3.2%, p < 0.0001) but significantly reduced viability of 33.4 ± 0.09% (p < 0.0001) compared to L3000. In NCI-H292, jetOPTIMUS yielded a lower transfection efficiency (22.6 ± 1.2%) with a significant reduction in viability (28.3 ± 0.9%, p < 0.0001). Other reagents varied significantly in their efficiency and impact on cellular viability in other cell lines. Changing the transfection mixture-containing medium at 6-hour post-transfection did not improve transfection efficiency or viability. However, pre-treatment of cell cultures with two rinses of 0.25% trypsin-EDTA improved transfection efficiency in 1HAEo- (85.2 ± 1.1% vs. 71.3 ± 1.0%, p = 0.004) and 16HBE14o- (62.6 ± 4.3 vs. 35.5 ± 1.2, p = 0.003).
Transfection efficiencies can differ based on airway epithelial cell line, reagents, and optimization techniques used. Consideration and optimization of cell line and transfection conditions may be useful for improving nonviral genetic techniques in vitro.
化学转染是气道上皮研究中广泛应用的技术，可用于研究基因表达变化及影响。此外，人们还探索了其在基因治疗递送方面的潜在应用。在此，我们对EX-EGFP-Lv105（一种表达增强绿色荧光蛋白的质粒）转染三种常用于模拟气道上皮的细胞系（1HAEo-、16HBE14o-和NCI-H292）的转染效率进行了表征。
我们使用了六种具有不同化学成分的常见和/或市售试剂：Lipofectamine 3000（L3000）、FuGENE HD、ViaFect、jetOPTIMUS、EndoFectin和磷酸钙。使用L3000时，与16HBE14o-和NCI-H292相比，1HAEo-表现出最高的转染效率（1HAEo-：76.1±3.2%，16HBE14o-：35.5±1.2%，NCI-H292：28.9±2.23%）。以对照组为标准，L3000产生了最高的转染效率，对细胞活力的影响最小，转染后48小时，1HAEo-细胞活力降低11.3±0.16%，16HBE14o-降低16.3±0.08%，NCI-H292降低17.5±0.09%。然而，jetOPTIMUS对1HAEo-的转染效率与之相似（90.7±4.2%，p = 0.94），但与L3000相比，其细胞活力显著降低，为37.4±0.11%（p < 0.0001）。在16HBE14o-细胞系中，jetOPTIMUS的转染效率显著高于L3000（64.6±3.2%，p < 0.0001），但与L3000相比，细胞活力显著降低，为33.4±0.09%（p < 0.0001）。在NCI-H292细胞系中，jetOPTIMUS的转染效率较低（22.6±1.2%），细胞活力显著降低（28.3±0.9%，p < 0.0001）。其他试剂在其他细胞系中的效率和对细胞活力的影响差异显著。转染后6小时更换含转染混合物的培养基并未提高转染效率或细胞活力。然而，用0.25%胰蛋白酶-EDTA冲洗细胞培养物两次进行预处理可提高1HAEo-（85.2±1.1%对71.3±1.0%，p = 0.004）和16HBE14o-（62.6±4.3对35.5±1.2，p = 0.003）的转染效率。
转染效率可能因气道上皮细胞系、试剂和所用的优化技术而异。考虑并优化细胞系和转染条件可能有助于改进体外非病毒基因技术。
