Systematic Optimization of 293T Cell Electroporation: Balancing High-Efficiency Gene Delivery with Cell Viability

Background: The goal of this study was to address the issues of low efficiency and poor cell survival during electroporation in 293T cells. The electroporation parameters were systematically optimized to improve gene delivery efficiency, cell viability, and reproducibility. Methods: Standard transfection protocols were refined via an orthogonal design approach combined with multiple detection assays. The "electroporation score" was used to evaluate the balance between transfection efficiency and cell viability. Results: Optimal conditions were identified (400 V, 500 μs, and 2 pulses), resulting in a transfection efficiency of (78.7 ± 3.1) % and maintaining cell viability at (79.0 ± 4.3) % in 1SM buffer. The electroporation score was highly effective in identifying parameter sets that balanced high efficiency with favorable survival. Discussion: The orthogonal design strategy successfully overcomes the limitations of conventional single-factor optimization. The electroporation score serves as a robust tool for the integrated assessment of electroporation outcomes. Furthermore, the optimized transfection protocol has improved the efficiency of subsequent experimental progress (such as obtaining fully positive cells, collecting engineered exosomes, etc.). Conclusion: An optimized, multidimensional protocol was established for the electroporation of 293T cells. This methodology also provides a standardized, scalable framework for gene editing in other cell types.