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

Systematic Optimization of 293T Cell Electroporation: Balancing High-Efficiency Gene Delivery with Cell Viability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Systematic Optimization of 293T Cell Electroporation: Balancing High-Efficiency Gene Delivery with Cell Viability Ming-Shi Zhang, Meng-Ru Li, De-Yun Zhang, Qiu-Yan Yin, Zhai-Zhuo Yu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8152999/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract 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. Biological sciences/Biological techniques Biological sciences/Biotechnology Physical sciences/Engineering Electroporation ATP assay CCK-8 Transfection efficiency Cell viability Electroporation score Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Advances in gene engineering and cell biology have underscored the importance of efficient and stable transfection technology for investigating gene function, protein expression, and cell signaling pathways. Electroporation offers a distinct advantage because of its wide applicability, high efficiency, and safety [1]. It is a physical method that achieves gene transfer by creating transient pores in cell membranes through electrical pulses. This physical method is suitable for a variety of cell types, particularly challenging ones such as primary cells, stem cells, and immune cells, which are difficult to transfect via conventional methods [2]. Electroporation is capable of delivering various molecules (DNA, RNA, miRNA, protein, etc.) effectively, with precise control over parameters and reproducible outcomes. Moreover, it circumvents the cytotoxicity associated with chemical reagents, the potential for insertional mutagenesis from viral vectors, and complex biosafety concerns. Consequently, electroporation has emerged as a robust and dependable technology pivotal for cell therapy, gene editing, and fundamental research [3]. The 293T cell line is widely used in gene expression research and virus packaging because of its immortalization ability and rapid proliferation [4]. The transfection efficiency of 293T cells can be significantly impacted by multiple factors, including the transfection buffer, parameters associated with transfection, and the cellular state. The current transfection efficiency of 293T cells is around 60%, which still has a significant gap compared to viral transfection. Therefore, systematically optimizing the electroporation protocol is essential for enhancing the experimental efficiency and reproducibility. In recent years, existing electroporation devices (such as the BTX ECM830 [5]) have gained widespread application in the field of cell transfection due to their ability to precisely control electroporation-related parameters. These can significantly improve cell transfection efficiency and survival rate by precisely controlling multiple parameters such as the electric field intensity, pulse duration, and pulse number. This study established an optimized transfection system for these cells on the basis of an orthogonal experimental design, integrating ATP detection, CCK-8 detection, and flow cytometer detection techniques: 1. Cellular ATP release assay: This assay quantifies the extracellular ATP concentration following electroporation, directly indicating the effects of various transfection parameters on transient membrane permeability [6](extent of pore opening), serving as a dynamic metric for parameter evaluation; 2. CCK-8 assay for cell viability analysis: Cells were co-incubated with the CCK-8 reagent for 1–2 hours following electroporation (24 hours post-transfection). The absorbance at 450 nm was measured via an ELISA reader to evaluate long-term survival rates and determine the damage threshold of the transfection parameters on the cell proliferation capacity. 3. Flow cytometry is employed to measure transfection efficiency accurately by assessing the expression levels of fluorescently labeled plasmids or reporter genes, such as GFP, in transfected cells. In this study, the transfection plasmid, derived from the GV712 plasmid, contains the EGFP gene GV712-EGFP, which is approximately 7.5 kP in length, enabling the exclusion of interference from cell debris or nonspecific signals. Furthermore, this study utilizes the "Electroporation score" (EPS) [7] as a comprehensive assessment tool. The EPS calculation formula combines cell viability data from CCK-8 assays with transfection efficiency data from flow cytometry, the EPS effectively represents the balance between "efficiency-viability" for various parameter combinations. This approach overcomes the constraints associated with optimizing a singular metric. This study utilized a multi-index linkage strategy to determine the optimal electroporation parameters (electric field voltage, pulse duration, and pulse number) for transfecting 293T cells via a BTX transfection instrument. This research not only identified the ideal parameter window but also elucidated the nonlinear correlation between cell membrane permeability (ATP release) and long-term activity (CCK-8 OD value). The established protocol offers a standardized approach for optimizing electroporation parameters for 293T cells. The modular design of the protocol, which includes single-factor analysis, orthogonal screening, CCK-8/flow detection, and EPS quantitative comparison, can be extrapolated to gene delivery studies involving challenging cell lines. This methodology provides a solid foundation for ensuring experimental reproducibility in gene editing, virus packaging, and related research fields. 2. Materials and methods 2.1 Materials 293T cells(ATCC; VA; USA), DMEM (biosharp; Cat. No. BL305A; Lot. No.00625678CJ; China), BTX buffer (BTXpress; CatalogNo.47 − 0002; Lot:4000079306, USA), Chica buffers(1S, 2S,1M, 2M, 1SM; For specific formulations, please refer to Table S1 in the supplementary materials), Electroporation cuvette (BTX, USA), ATP chemiluminescence assay kit (Servicebio; Cat:G4309-48T; Lot:MPC2409061; China), Cell Counting Kit-8 (biosharp; Cat. No. BS350B; Lot: 23360953; China), PBS (1×) (SparkJade; REF: CR0014-500ML; LOT: FFELY; China), Fetal bovine serum standard (Cell max; Cat No. SA301.02. V;lot No.20221220; China), 0.25% Trypsin-EDTA(1×)(GOONIE༛Cat: G100-502༛Lot༚250322; China), Penicillin/Streptomycin(100×)(Goonie; Cat༚100–503; Lot༚LGM241204; China), Flow Cytometer(FACSVerse; BD; USA), Synergy Neo2 Microplate Reader(BioTek; USA༉, ECM830 Square Wave Electrical Converter(BTX; USA), Sorting Flow Cytometer (FACSAria III; BD; USA), GV712-EGFP Plasmid(GENECHEN; China). 2.2 Methods 2.2.1 Ethics Approval The 293T cell line used in this study was purchased from the American Type Culture Collection (ATCC) through official laboratory channels, and the source was compliant. This cell line is a commercial immortalized cell line. According to the current ethical standards at home and abroad, such research is usually exempt from ethical review. This study is an in vitro cell experiment that does not involve human embryos or clinical applications and meets the ethical requirements of biomedical research. 2.2.2 Plasmids and Cloning The GV712-EGFP plasmid used in this experiment was synthesized and provided by Jikai Gene Co., Ltd. Using the GV712 plasmid as the design vector, the plasmid was cut after the CMV promoter protein gene with XbaI enzyme, and then a gene fragment containing Glycosylation sequence protein GNSTM with-3-EGFP-3FLAG was inserted to construct (-CMV-GNSTM-3-EGFP-3FLAG-puromycin-). 2.2.3 Cell culture 293T cells were cultured in complete DMEM (containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin double antibody) in a constant-temperature incubator at 37°C and 5% CO 2 [ 8 ]. Upon reaching the logarithmic growth phase (approximately 80–90% confluence), the old culture medium was aspirated, and the cell monolayer was gently rinsed with phosphate-buffered saline (PBS). An appropriate volume of trypsin solution was then applied to dissociate the adherent cells. After complete digestion, the trypsin activity was neutralized by adding complete medium, and a single-cell suspension was obtained by gentle trituration. The cell suspension was subsequently centrifuged to form a pellet, followed by cell counting. The cells were then either passaged at a suitable ratio or subjected to a second centrifugation and resuspended in electroporation buffer at an optimal density for subsequent electroporation experiments. 2.2.4 Electroporation and optimization scheme Using a single-factor design to control variables, a specific high-efficiency buffer with the highest transfection efficiency and best protective effect for 293T cells was screened from seven electroporation buffers. Subsequently, the maximum tolerable ranges for pulse voltage, pulse duration, and pulse number in 293T cells were sequentially determined using the ATP assay. Under the selected specific buffer conditions, appropriate levels were chosen from the tolerance ranges of the three parameters for subsequent orthogonal design experiments. (1) Single-factor design 1) Selection of cell-specific high-efficiency buffer 293T cells at the logarithmic growth phase (approximately 80% confluence) were harvested via trypsinization, collected by centrifugation, and counted via a hemocytometer. The cells were resuspended in the respective buffers—DMEM, BTX buffer, and Chica buffers [ 9 ] (including 1 SM, 1 S, 2 S, 1 M, and 2 M)—at a density of 5×10⁶ cells/mL. The corresponding amount of GV712-EGFP plasmid (5 µg per 400 µL of cell suspension) was added to each group. A volume of 400 µL of the mixture was gently transferred into a 2 mm electroporation cuvette, and the cuvette was tapped gently to eliminate air bubbles. Transfection was performed via identical parameters for all groups, with each condition tested in triplicate. The data are presented as the means of three replicates. To compare the transfection efficiency across the different electroporation buffers, one-way analysis of variance (ANOVA) was conducted. If significant intergroup differences were detected, Tukey test was applied for multiple comparisons. All the statistical analyses were performed via GraphPad Prism (version 9.5.0) software. 2) Optimization of electroporation parameters The extent of cell membrane permeabilization under a given set of electroporation parameters was determined by measuring the amount of adenosine triphosphate (ATP) released extracellularly immediately following electric pulse delivery, according to the ATP content assay described by Marie-Pierre et al. [ 10 ]. This assay indirectly reflects the degree of pore formation in the plasma membrane by quantifying the efflux of intracellular ATP molecules, thereby helping to establish tolerable ranges for key parameters: voltage, pulse duration, and pulse number. Electric field strength is the critical determinant of successful membrane permeabilization [ 11 ]. Owing to the fluid nature of the phospholipid bilayer, an electric field strength below a specific critical threshold (CT) fails to effectively disrupt membrane integrity, preventing significant ATP release. Conversely, when the field strength exceeds a certain highest threshold (HT), excessive energy causes irreparable damage to the phospholipid bilayer, leading to cell rupture and death—a process known as irreversible electroporation [ 12 ]. Pulse duration and pulse number are essential parameters for sustaining membrane pores. Once pores are successfully created by an applied electric field, the inherent properties of the membrane drive rapid resealing. An appropriate pulse duration and number help maintain the existence and quantity of pores in the phospholipid bilayer without compromising cell viability, thereby facilitating the release of intracellular ATP molecules. On the basis of the aforementioned principles, a controlled variable approach was employed. Under constant conditions, experiments were designed with gradient increases in the transfection voltage, pulse duration, and pulse frequency. The specific experimental design is detailed in Table 1 . Following electroporation, the cells were removed under appropriate centrifugation conditions, and the supernatant was collected. Detection was performed via a chemiluminescent ATP assay kit, and the corresponding ATP release curves were plotted. Table 1 Single-Factor Analysis Design Factor Tested Fixed Conditions Buffer All other transfection parameters held constant Voltage Pulse duration: 100 µs, number of pulses: 3, and all other transfection parameters held constant Pulse Duration Voltage: 300V, number of pulses: 3, and all other transfection parameters held constant Number of Pulses Voltage: 300V, pulse duration: 100 µs, and all other transfection parameters held constant (2) Orthogonal design On the basis of the three ATP content detection curves plotted from the single-factor analysis, three suitable parameter levels were selected, and a three-factor, three-level orthogonal experimental design (L9 (3³)) was conducted, as detailed in Table 2 . The results of the orthogonal experiments were compared through range analysis and analysis of variance to determine the optimal combination of the three transfection parameters for validation. Table 2 − 1: Orthogonal design Level Factor A: Voltage (V) B: Duration (µs) C: Number of Pulses 1 300 100 2 2 350 300 4 3 400 500 6 Table 2 2: Orthogonal design experiment groups No. A B C 1 1 1 1 2 1 2 2 3 1 3 3 4 2 1 2 5 2 2 3 6 2 3 1 7 3 1 3 8 3 2 1 9 3 3 2 2.3 Calculation of Electroporation Scores 2.3.1 Assessment of cell viability via the CCK-8 assay Following an orthogonal design, the cells were electrotransfected via the ECM830 square wave electrical converter. After electroporation, the electrode cups were allowed to stand at room temperature for 10–15 minutes. The cells were then slowly resuspended via a dedicated Pasteur pipette to ensure uniform dispersion. A suspension of 2 × 10⁴ cells from each group was seeded into 96-well plates containing fresh medium prewarmed to 37°C in a water bath, with three parallel wells per group. After all groups were seeded, the plates were incubated at 37°C for 24 hours. Following the instructions of the CCK-8 assay kit, the absorbance at 450 nm was measured for each transfected cell group via a Synergy Neo2 microplate reader. The specific workflow is illustrated in Fig. 1 . The data were processed via GraphPad Prism 9.5.0 software. Cell viability was calculated as follows: Cell viability(%) = (OD transfection experimental – OD control medium group)/(OD blank cell – OD control medium group)×100%. Dunnett's test in one-way ANOVA was performed between each group and the blank cell group to assess statistical significance (P 0.05). 2.3.2 Flow cytometry detection of EGFP fluorescent signal The uniformly resuspended electroporated cells were transferred by group into six-well plates containing fresh medium prewarmed to 37°C. After 24 hours of culture, the cell status was observed, and a half-medium change was performed. The cells were further cultured for approximately 72 hours, when they had largely recovered and reached an appropriate density. The cells were then trypsinized, collected, and resuspended in PBS (300 µl) to prepare single-cell suspensions. Transfection efficiency was assessed via a flow cytometer. Data analysis was performed via Flow Jo software (version 10.8.1). Statistical analysis was conducted via one-way ANOVA followed by Tukey test via GraphPad Prism software (version 9.5.0). 2.3.3 Calculation of electroporation score To synthesize the effects of transfection parameters, the Electroporation Score (EPS) concept proposed by Chicaybam L [ 6 ] in 2017 was specifically applied. EPS was calculated as: EPS = Cell Viability (%) × Expression (%) / F, where the fitting factor F was 50 for adherent cell lines and 100 for non-adherent cell lines. This adjustment facilitated graphical representation and provided a more comprehensive and intuitive quantification of transfection outcomes. 3. Results 3.1 Determination of an efficient buffer As shown in Fig. 2 , under identical electroporation conditions, different buffers exhibited significantly different transfection efficiencies. Among them, buffers 1 SM, 2 S, and 1 S demonstrated superior transfection efficiency compared with other buffers, with 1 SM buffer achieving the highest transfection efficiency. 3.2 Cellular ATP release curve On the basis of the single-factor analysis principle described in Section 2.2.2 , with a fixed pulse duration of 100 µs and three pulses administered, the pulse voltage was set in a gradient. Following electroporation, the cell suspensions were collected from each group of electroporation cuvettes and centrifuged at 1000 rpm for 3 minutes, and the supernatants from each group were obtained. The supernatants were transferred into white opaque 96-well plates (with three replicate wells per group). The prepared ATP detection working solution was mixed with the supernatants according to the protocol of the ATP detection kit (Sevier). Luminescence values were then rapidly measured for each well via a chemiluminescence-detecting Synergy Neo2 microplate reader. As shown in Fig. 3 -A, when the electric field strength exceeded 0.25 kV/cm (corresponding to an electroporation voltage greater than 50 V), the electric shock successfully punctured the cell membrane, and intracellular ATP began to be released into the external environment. This electric field strength represents the critical threshold (CT) for cell membrane opening. As the electric field strength continued to increase, the extent of membrane opening also increased. When the electric field strength increased from 2.0 kV/cm to 2.5 kV/cm, the amount of ATP released by the cells reached a maximum plateau. Observation of the cells after transfection revealed that the electric field intensity at this point caused irreversible damage to the cell membrane, ultimately leading to cell rupture and death. Therefore, 2.5 kV/cm was determined to be the highest threshold (HT) for membrane opening that the cells could withstand. On the basis of these findings, the electric field strength levels selected for the orthogonal design were 1.5 kV/cm, 1.75 kV/cm, and 2.0 kV/cm. Following the same protocol, a fixed electric field strength of 1.5 kV/cm was selected from the range of tolerable electric field strengths determined experimentally. As shown in Fig. 3 -A, the voltage applied at this point effectively opened the cell membrane while maintaining cellular viability with minimal impact. The pulse count was held constant at 3 pulses, while the pulse duration was increased in a gradient manner. The ATP release levels were then measured across each group to determine the range of pulse durations the cells could tolerate, which provided an appropriate design range for subsequent orthogonal experiments. The final results are presented in Fig. 3 -B. On the basis of these experimental results, the pulse duration levels selected for the orthogonal design were 100 µs, 300 µs, and 500 µs. The experimental method for pulse frequency was identical to the two aforementioned detection methods, with the final results presented in Fig. 3 -C. After comprehensive consideration of the orthogonal design, the frequency levels were ultimately selected as 2, 4, and 6. 3.3 Orthogonal Design 3.3.1 Selection of electric field intensity, pulse time, and pulse number gradient On the basis of the optimal factor levels identified through single-factor analysis, the experiment was designed according to the orthogonal design table with three factors at three levels (L9 (3³)). The detailed experimental parameters are presented in Table 3 . 3.3.2 Transfection efficiency detection Following the method described in Section 2.3.2 , cells that had recovered from the confluent state after transfection were digested and harvested. After resuspension in PBS buffer, the transfection efficiency of cells in each orthogonal group was measured via a flow cytometer (BD FACS Verse). Each group was analyzed in triplicate, and the average value was recorded. The flow cytometry results for the selected groups in the orthogonal experiments are shown in Fig. 4 . The transfection efficiency results for all the experimental groups are summarized in Fig. 5 . 3.3.3 Cell viability in each group Following the assay method described in Section 3.1 , cells from each group subjected to electroporation were seeded into 96-well plates for culture, with three parallel wells per group. Cell viability was assessed via a CCK-8 assay. The mean viability across the three wells per group served as the final viability result for that group. The viability of the blank group was set as 1.00. All other groups were normalized to the blank group for comparison, and the results are shown in Fig. 6 . Compared with the blank group, Groups 4, 6 and 8 did not significantly differ in viability across groups(ns), indicating that the transfection parameters used in Group 6 did not cause irreversible substantial damage to the cells themselves. For the transfection parameters of the other groups, the cells were affected to varying degrees, particularly in Group 9, where cell viability was significantly impacted (****) by the corresponding parameters. 3.4 Electroporation scoring and orthogonal experimental results validation The transfection efficiencies and cell viabilities of the nine orthogonal cell groups, obtained via flow cytometry and CCK-8 assays, respectively, were determined. The electroporation scores for each group were calculated via the formula outlined in Section 2.3.3 (Fig. 7 , Table 3 − 1). On the basis of the electroporation scores, the range method and variance method from the orthogonal experiments were applied to analyze and determine the optimal levels of the three factors within the orthogonal design. Table 3 − 1: Orthogonal design experiment results No. A B C Expression(%) (Mean ± SD) Viability(%) (Mean ± SD) EPS(%) 1 1 1 1 7.4 ± 0.8 85.5 ± 2.8 12.65 2 1 2 2 28.9 ± 2.8 84.9 ± 8.7 49.07 3 1 3 3 63.3 ± 3.9 55.9 ± 4.9 70.76 4 2 1 2 23.9 ± 1.3 92.9 ± 2.5 44.41 5 2 2 3 34.0 ± 5.7 53.5 ± 2.8 36.38 6 2 3 1 50.5 ± 1.8 93.6 ± 6.3 94.54 7 3 1 3 33.8 ± 3.4 70.1 ± 8.7 47.38 8 3 2 1 47.8 ± 2.5 91.3 ± 1.6 87.28 9 3 3 2 78.5 ± 3.5 36.5 ± 3.3 57.31 Table 3 − 2: Range analysis Range Analysis A B C K1 44.160 33.813 64.823 K2 58.443 57.577 50.263 K3 63.990 74.203 51.507 R 19.830 38.390 14.560 Table 3 3: Analysis of Variance Analysis of Variance Sum of Squares (SS) Degrees of Freedom (df) F A 628.008 2 0.560 B 2346.187 2 2.092 C 390.873 2 0.348 Error 3365.07 6 Through analysis of the range (Table 3 − 2) and variance (Table 3 – 3 ) of the orthogonal design experiment results, the levels with the highest K values among the three factors A, B, and C were A3, B3, and C1, respectively. Thus, the optimal theoretical combination for 293T cells within this orthogonal range was A3B3C1, corresponding to transfection parameters of 2.0 kV/cm, 500 µs, and 2 pulses. Among these factors, factor B presented the highest R value, indicating that pulse duration was the most influential factor affecting cell transfection in this orthogonal design. The analysis of variance further corroborated this conclusion. According to the results of the orthogonal design experiment, a validation group was designed using the following transfection parameters: 400 V, 500 µs, and 2 pulses, with all other factors held constant for the transfection of 293T cells. The final results of three replicate experiments were shown in Fig. 8 . While ensuring that cell viability was no lower than 70.0%, the transfection efficiency reached (78.7 ± 3.1) %, with a score of 124.3. This score exceeded those of all orthogonal groups. Compared with the groups shown in Fig. 6 , the validation group successfully enhanced the transfection efficiency while maintaining cell viability. The results met expectations, confirming the validity of this orthogonal design. 3.5 Positive Cell Sorting The 293T cells were transfected using the optimized transfection parameters obtained through orthogonal design and a multi-index combined system. After reaching a stable state, the cells were digested, collected, and resuspended in complete medium to form a single-cell suspension at a concentration of 1.0 × 10 6 cells/ml. Using a sorting flow cytometer, positive cells expressing EGFP protein were sorted under the 488 nm excitation channel, ultimately yielding highly positive cells (as shown in Fig. 9 ). After 30 days of continuous culture, the sorted cell population maintained stable growth and exhibited sustained, uniform EGFP fluorescence. 4. Discussion 4.1 Combinations of Multiple Methods This study adopted an orthogonal design as the research approach, integrating multiple indicator methods such as the ATP assay and the CCK-8 assay to establish an efficient and systematic electroporation condition optimization system. This approach provides a reliable solution for rapidly screening optimal parameter combinations that balance transfection efficiency and cell viability, effectively overcoming the challenges inherent in traditional electroporation condition optimization. These challenges include reliance on single qualitative indicators, low transfection efficiency, high experimental blindness, and difficulty in simultaneously addressing membrane permeability and cell viability. Orthogonal design represents a highly efficient multifactor optimization strategy widely applied in fields process, formulation, or parameter optimization [ 13 – 14 ]. Its core advantage lies in scientifically and efficiently analyzing and determining optimal combinations within multifactor, multilevel problems with minimal experimental runs. These strengths perfectly align with the objectives of optimizing electroporation experiments for cells. Among the numerous factors involved in cell electroporation experiments, the three most critical factors [ 15 ] are: (1) electric field, whether it can successfully "open" the cell membrane without compromising subsequent cell viability. A low electric field failed to effectively puncture the cell membrane, whereas a high electric field caused irreparable damage to the cell membrane [ 16 ]; (2) time and number of pulses, short durations or few pulses failed to sustain the "channel" opened by the electric field, whereas prolonged durations or excessive pulses resulted in prolonged membrane opening and excessive shock, leading to cell death. However, since different cell lines exhibit varying tolerances to electroporation, confirming the key transfection factors and rationally selecting their parameter levels are crucial in orthogonal design. The ATP assay served as a suitable solution. The amount of ATP released by electroporated cells reflects the extent of membrane opening. This method allowed for the gradual determination of tolerable ranges for the three factors, providing objective and reasonable basis for selecting the level gradients of each factor. The use of the CCK-8 assay to assess cell viability post transfection not only quantified cellular recovery following electroporation but also, when analyzed alongside ATP detection curves, revealed the nonlinear relationship between membrane permeability and long-term viability. Specifically, increased membrane permeability was correlated with greater ATP release, whereas conversely, cellular activity decreased. 4.2 Electroporation score The evaluation of a transfection parameter's effect on electroporated cells involves two main aspects: the expression rate of transfected cells and their subsequent viability [ 17 ]. The primary objective of this study was to identify the optimal overall performance of the transfection parameters through various detection methods rather than pursuing the highest expression rate or highest survival rate in isolation. Therefore, to avoid the accuracy of the final results being affected by an extreme positive or negative value of a single parameter, as shown in Fig. 7 , when considering only the cell expression rate, the expression rate of orthogonal group 9 reached 78.5 ± 3.5% (Fig. 5 ), which was higher than that of the other experimental groups and the control group. However, the cell viability in this group was significantly affected by the transfection parameters, with cell viability less than 40% (Fig. 6 ). Therefore, a comprehensive evaluation integrating both aspects was essential to achieve the ultimate objective of this study. The EPS serves as a comprehensive quantitative metric capable of rationally converting two or more evaluation indicators. Through computational methods (as outlined in Section 2.3.3 ), a single score was generated to holistically assess the "quality" of an electroporation experiment. Through simple mathematical operations, it integrates transfection efficiency and cell viability—two metrics that often constrain each other—transforming complex multi objective trade-offs into an intuitive composite score. This approach enabled clear quantification and comparison of the equilibrium between transfection efficiency and cell viability under different parameter conditions. Subsequent analyses could then precisely determine the extent to which specific transfection parameters impact the overall cellular state. 4.3 Analysis of the orthogonal design experiment results Drawing on the range and variance analysis of the orthogonal design experiment results (Tables 3 − 2 and 3–3), within the designed orthogonal range, pulse duration (factor B) was the primary factor influencing EPS (R = 38.390, F = 2.092), with its significance far exceeding that of electric field voltage (A) and pulse frequency (C). This finding provides crucial insight: pulse duration directly determines the duration of cell transfection, thereby influencing the influx of exogenous substances and the self-repair process of the cell membrane. Insufficient duration resulted in inadequate delivery of the target plasmid, whereas excessive duration caused irreversible membrane damage and imbalances in ions and other substances, ultimately leading to cell death. The optimal parameter combination A3B3C1 (400 V, 500 µs, 2 pulses) achieved a balance between high transfection efficiency and high cell viability precisely because it identified the optimal equilibrium between the membrane permeability window and cellular tolerance. This further demonstrated the comprehensiveness and rationality of using EPS as an orthogonal design experiment result evaluation metrics. It also demonstrated the rationality of this optimization system that combines orthogonal design with multiple indicators. 4.4 Acquisition of Fully Positive Cells Highly efficient and stable transfection meant that more cells successfully incorporated and expressed the target gene (EGFP), thereby forming a high proportion of positive cell populations within the cell population. This advantage ensured that when using a sorting-type flow cytometer for positive cell sorting in subsequent steps, there was an adequate number of target cells with clear signals, significantly simplifying the sorting process, reducing sorting time, and lowering the experimental time and resource costs. Therefore, this optimized scheme not only improved the transfection efficiency itself but also provided technical support for the subsequent efficient acquisition of fully positive cell clones, demonstrating the significant gain in overall experimental workflow efficiency through systematic optimization. 4.5 Research limitations This study has several limitations, too. First, all the optimization work was conducted using the 293T cell line. Although this approach has shown theoretical potential for extension to other difficult-to-transfect cell types, further validation is needed. Second, the study indirectly assessed membrane permeability through ATP release but did not investigate the dynamic mechanisms of post electroporation membrane repair or cell death pathways. As noted by Kotnik et al. [ 18 ], the mechanisms of membrane resealing after electroporation and their relationship with cell survival remain significant challenges in the field. Future studies can employ molecular dynamics simulations combined with real-time imaging techniques to further elucidate these dynamic processes. Additionally, the optimal parameters identified in this protocol were specific to a particular electroporation device (ECM830; BTX; USA), and their applicability to other instruments might be limited owing to differences in electric field design. Future studies can verify this scheme in more cell models and further deepen the applicability depth and reliability of this optimized system by combining membrane repair mechanism research with functional endpoint indicators. 5. Conclusion This study systematically optimized key parameters for electroporation transfection in 293T cells and established an efficient screening system for transfection conditions on the basis of an orthogonal experimental design and multi-indicator integration. ATP release assays define the dynamic range of changes in cell membrane permeability. The CCK-8 assay and flow cytometry were employed to evaluate long-term survival rates and transfection efficiency in transfected cells, respectively. The introduction of EPS serves as a comprehensive metric that effectively balances the trade-off between transfection efficiency and cell viability. The experimental results demonstrated that under optimized electroporation buffer (1 SM buffer) and the theoretically optimal parameter combination (400 V, 500 µs, 2 pulses), both the transfection efficiency and cell viability in 293T cells were significantly enhanced, resulting in the highest EPS score. This finding validates the effectiveness and reliability of the orthogonal design and multi-indicator integration strategy. Furthermore, it was also significantly improved the efficiency of subsequent flow cytometric sorting to obtain fully positive cells, making the process more convenient and efficient, and greatly saving time and experimental costs. This study provides not only a standardized, reproducible protocol for electroporation of 293T cells but also a modular optimization strategy that can be extended to other cell types. It offers methodological support and a reliable optimization plan for research in gene editing and cell therapy. Declarations Acknowledgements Not applicable. Author contributions Conceptualization: M.S.Z, B.Y, Z.Y.Z. Data curation and Formal analysis: M.S.Z, M.R.L, Q.T.Z, B.Y. and Z.Y.Z. ATP detection: M.S.Z, M.R.L, D.Y.Z, Q.Y.Y. and Z.Z.Y. Flow Cytometry Analysis: M.S.Z, M.R.L, Z.Y.Z. Funding acquisition: B.Y. Resources: M.S.Z, B.Y, Z.Y.Z. Supervision: Z.B.G, J.X.W, N.P.Z and F.F.C. Writing—original draft: M.S.Z. Writing—review & editing: Q.T.Z, B.Y, Z.Y.Z. Funding Not applicable. Availability of data and materials All data used during the current study available from the corresponding author on reasonable request. Ethics approval and consent to participate. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Department of Traditional Chinese Pharmacy, School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. 2 Department of Pharmacology, Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. 3 Department of Traditional Chinese Pharmacy, Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. 4 Department of Neurosurgery, Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. 5 Department of Traditional Chinese Pharmacy, School of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. 6 Department of Medical Immunology, School of Clinical and Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong. References Sun, H., Yu, L., Chen, Y., Yang, H. & Sun, L. Analysis of In Situ Electroporation Utilizing Induced Electric Field at a Wireless Janus Microelectrode. Micromachines (Basel) . 15 (7), 819 (2024). Sioud, M. Optimized siRNA Delivery into Primary Immune Cells Using Electroporation. Methods Mol. Biol. 2115 , 119–131 (2020). Liu, F. et al. Advanced micro/nano-electroporation for gene therapy: recent advances and future outlook. Nanoscale 16 (22), 10500–10521 (2024). Wang, C. et al. Engineering a HEK-293T exosome-based delivery platform for efficient tumor-targeting chemotherapy/internal irradiation combination therapy. J. Nanobiotechnol. 20 (1), 247 (2022). McIntyre, D. A. & Harlander, S. K. Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. Appl. Environ. Microbiol. 55 (3), 604–610 (1989). Gibot, L. et al. Calcium Delivery by Electroporation Induces In Vitro Cell Death through Mitochondrial Dysfunction without DNA Damages. Cancers (Basel) . 12 (2), 425 (2020). Chicaybam, L. et al. An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells. Front. Bioeng. Biotechnol. 4 , 99 (2017). Li, X. L. et al. Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression. Nucleic Acids Res. 46 (19), 10195–10215 (2018). Chicaybam, L., Sodre, A. L., Curzio, B. A. & Bonamino, M. H. An efficient low cost method for gene transfer to T lymphocytes. PLoS One . 8 (3), e60298 (2013). Rols, M. P. & Teissié, J. Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. Biophys. J. 58 (5), 1089–1098 (1990). VanderBurgh, J. A., Corso, T. N., Levy, S. L. & Craighead, H. G. Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing. Sci. Rep. 13 (1), 6857 (2023). Zhang, N. et al. l. Irreversible Electroporation: An Emerging Immunomodulatory Therapy on Solid Tumors. Front Immunol. ;12:811726. (2022). Luna, J., Jaynes, J., Xu, H. & Wong, W. K. Orthogonal array composite designs for drug combination experiments with applications for tuberculosis. Stat. Med. 41 (17), 3380–3397 (2022). Zhu, C. et al. Optimizing glycerosome formulations via an orthogonal experimental design to enhance transdermal triptolide delivery. Acta Pharm. 72 (1), 135–146 (2021). Campillo-Davo, D. et al. The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy. Pharmaceutics 13 (3), 396 (2021). Campelo, S. N., Huang, P. H., Buie, C. R. & Davalos, R. V. Recent Advancements in Electroporation Technologies: From Bench to Clinic. Annu. Rev. Biomed. Eng. 25 , 77–100 (2023). Timmins, L. M. et al. Selecting a Cell Engineering Methodology During Cell Therapy Product Development. Cell. Transpl. 30 , 9636897211003022 (2021). Kotnik, T., Rems, L., Tarek, M. & Miklavčič, D. Membrane Electroporation and Electropermeabilization: Mechanisms and Models. Annu. Rev. Biophys. 48 , 63–91 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 20 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviews received at journal 23 Dec, 2025 Reviewers agreed at journal 03 Dec, 2025 Reviewers invited by journal 28 Nov, 2025 Editor invited by journal 25 Nov, 2025 Editor assigned by journal 20 Nov, 2025 Submission checks completed at journal 20 Nov, 2025 First submitted to journal 19 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8152999","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":552814759,"identity":"d8bb9591-d920-4f63-bc94-3313dab2d77f","order_by":0,"name":"Ming-Shi Zhang","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ming-Shi","middleName":"","lastName":"Zhang","suffix":""},{"id":552814760,"identity":"0c6a968b-2155-409a-9249-f216e88ad9d4","order_by":1,"name":"Meng-Ru Li","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Meng-Ru","middleName":"","lastName":"Li","suffix":""},{"id":552814761,"identity":"d5df94c2-0a1c-48ec-ab7f-ccfec5bcc80f","order_by":2,"name":"De-Yun Zhang","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"De-Yun","middleName":"","lastName":"Zhang","suffix":""},{"id":552814762,"identity":"227a3a14-5f0c-422e-ac8e-dc6200fe6d5b","order_by":3,"name":"Qiu-Yan Yin","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qiu-Yan","middleName":"","lastName":"Yin","suffix":""},{"id":552814763,"identity":"39c2b926-7a91-4734-ad37-484c7e11f1d9","order_by":4,"name":"Zhai-Zhuo Yu","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhai-Zhuo","middleName":"","lastName":"Yu","suffix":""},{"id":552814764,"identity":"b2112267-b228-470a-8de0-4a9f6a0dc810","order_by":5,"name":"Zhi-Bo Gai","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Bo","middleName":"","lastName":"Gai","suffix":""},{"id":552814765,"identity":"259a6834-cdde-4a2e-9c27-0cb394d9f929","order_by":6,"name":"Jun-Xiang Wang","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jun-Xiang","middleName":"","lastName":"Wang","suffix":""},{"id":552814766,"identity":"ca96d6d3-27aa-4a00-b72c-7325bff132e8","order_by":7,"name":"Nian-Ping Zhang","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nian-Ping","middleName":"","lastName":"Zhang","suffix":""},{"id":552814767,"identity":"d8df209e-6d75-445b-bd47-d36440a35a9c","order_by":8,"name":"Fen-Fen Chen","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fen-Fen","middleName":"","lastName":"Chen","suffix":""},{"id":552814768,"identity":"b09bd602-2d71-4c58-bb75-fa224841e3e2","order_by":9,"name":"Qi-Tao Zhao","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qi-Tao","middleName":"","lastName":"Zhao","suffix":""},{"id":552814769,"identity":"d8b16e43-846e-4a6c-9ddd-375ddb181d8d","order_by":10,"name":"Zhi-Yong Zhang","email":"","orcid":"","institution":"Shandong First Medical University\u0026 Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Yong","middleName":"","lastName":"Zhang","suffix":""},{"id":552814770,"identity":"309220e0-b91c-4395-8b61-da2ddfde1cbb","order_by":11,"name":"Bin Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFACHoYDIIqfmbHxwQcDGzvitUi2Nx82nFGQlkyUFjAwOHMsTZrnwyHGBkIazNt7Dx4ubLPLY7iRYyBtY3CAmYH98NEN+LTInDmXcHhmW3Ix44wcA+Mcgzt8DDxpaTfwaZGQyDE4zNvGnNgMZCTnGDxjZpDgMSNGS31iG4hhYXCYsYFILYcTe3iOJTYzEKWFB+gXnnPHE2ewNx9m7DFIS2Yj6Bf23sOfecqqE/cfZmz/8eOPjR0/++FjeLWAASMbEocNpzIU8Ic4ZaNgFIyCUTBCAQCcRUzCa4ge4QAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2025-11-19 08:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8152999/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8152999/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97134073,"identity":"34db6e98-98a6-4696-bc37-f264941cbf81","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2253616,"visible":true,"origin":"","legend":"","description":"","filename":"SystematicOptimizationof293TCellElectroporationBalancingHighEfficiencyGeneDeliverywithCellViability.docx","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/1df643667c21eddd693b4796.docx"},{"id":97142760,"identity":"c1e0f130-5967-443d-a8a6-39ba101c88d2","added_by":"auto","created_at":"2025-12-01 10:07:56","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11890,"visible":true,"origin":"","legend":"","description":"","filename":"c282ea4e79c74849b5ad8515a3cdd43e.json","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/cf9e926d30980eb336511b4e.json"},{"id":97142530,"identity":"ac9752e0-5f84-42f5-a4d0-c332481de152","added_by":"auto","created_at":"2025-12-01 10:07:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":877778,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/3fe28d71616b2de2a649b99d.docx"},{"id":97143003,"identity":"5a865274-64ed-48f2-bf80-75335be1b60c","added_by":"auto","created_at":"2025-12-01 10:08:11","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103364,"visible":true,"origin":"","legend":"","description":"","filename":"c282ea4e79c74849b5ad8515a3cdd43e1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/a768bcff56d5e7a9ad89fdf4.xml"},{"id":97134058,"identity":"73e85fed-c74b-400f-8d6b-d05d77a8b370","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":329780,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/613b5a21d54ffb5340605067.jpeg"},{"id":97134057,"identity":"1fc45cb8-5e88-40b4-b1d2-e3e10d9b9c2a","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68491,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/f61c3ba3f1fe4d2ebea235a5.jpeg"},{"id":97134061,"identity":"a8b91b6a-8d80-46fd-a0af-7f8e132d1660","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166814,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/12bcb78e9e7933226e5ffeb6.jpeg"},{"id":97134064,"identity":"c5d51de7-a525-490b-8def-d0cd1819c73c","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138168,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/d518b666bbc0114bbd54f2b4.jpeg"},{"id":97134066,"identity":"899465f9-3546-4efb-a0f7-19dee0050063","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61324,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/a926238b35e838897b3fe4a3.jpeg"},{"id":97141906,"identity":"cdf5d3c3-af0c-4a51-b707-2a25da46002b","added_by":"auto","created_at":"2025-12-01 10:07:09","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94099,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/28d5327cd3b68fc1a6f188f5.jpeg"},{"id":97142387,"identity":"71fd2f2d-b871-4498-94db-9d1a52bfd137","added_by":"auto","created_at":"2025-12-01 10:07:34","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":309813,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/967e7d4b28f4c1e28e58acb8.jpeg"},{"id":97141833,"identity":"6d3da2dd-fcc0-4da6-9ab7-8d33c0dbac43","added_by":"auto","created_at":"2025-12-01 10:07:05","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176643,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/6319c4aa619cd891653d966d.jpeg"},{"id":97134076,"identity":"4a0edd5e-29e0-490e-aba0-a5d962825d68","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":833119,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/5def7d417a4da506a439e47b.jpeg"},{"id":97142474,"identity":"63f63764-72b3-48d5-8647-564b2b52957b","added_by":"auto","created_at":"2025-12-01 10:07:38","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87516,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/0248bde64af81fcc82f2cff2.png"},{"id":97142563,"identity":"50804ec6-e599-4d79-8cae-dc1b9da0908a","added_by":"auto","created_at":"2025-12-01 10:07:43","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14611,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/583117d0a501ac0425f65dc2.png"},{"id":97142963,"identity":"80fa6ada-573f-43f6-9b3e-83dfb33cafaf","added_by":"auto","created_at":"2025-12-01 10:08:09","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94706,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/baf747ee0b59cae5ee3799ac.png"},{"id":97142964,"identity":"42f2f9ce-c373-4c39-81bc-15d5d7f81623","added_by":"auto","created_at":"2025-12-01 10:08:09","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59547,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/537a6f0819a02981aad61ad0.png"},{"id":97134069,"identity":"8f91b431-c88c-4572-a7d8-7315bcf3f0fd","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10319,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/1fafa93ca2f7127cd3248524.png"},{"id":97142210,"identity":"1b3fd252-c473-4a67-b7a3-d490c98b5b27","added_by":"auto","created_at":"2025-12-01 10:07:25","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13655,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/06d4703ad7b2dd1650cc2d4c.png"},{"id":97134078,"identity":"2fabe46a-cd5b-4b07-a043-8d8962623aa2","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44289,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/435f50af00f509f4096e1852.png"},{"id":97134079,"identity":"05866b8c-2abc-487d-a80b-2f73a912a903","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43265,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/b443a3ab2949c94ee500966a.png"},{"id":97134081,"identity":"12840759-01d9-4a19-93d2-17949741b061","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":283810,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/97d1b9759a4967b3ca5571fe.png"},{"id":97134082,"identity":"952f0f59-95ca-4048-950d-e8a1e24f3077","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102596,"visible":true,"origin":"","legend":"","description":"","filename":"c282ea4e79c74849b5ad8515a3cdd43e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/bd3654f7a8424a70d9f52cae.xml"},{"id":97142531,"identity":"3111e5da-b626-41bb-bb55-c100205b5e6d","added_by":"auto","created_at":"2025-12-01 10:07:42","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114461,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/801d203af718d0984dbb0daa.html"},{"id":97134050,"identity":"95097ec8-12a7-4a57-a35e-6e1abf1e4b61","added_by":"auto","created_at":"2025-12-01 09:13:51","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329780,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the cell manipulation workflow. ①Transfection efficiency assay: Transfected cells were seeded into 6-well plates containing prewarmed fresh complete medium and cultured for 72 hours. Transfection efficiency was then assessed via flow cytometry. ②Cell viability assay: Transfected cells were seeded into 96-well plates and cultured for 24 hours. After CCK-8 treatment, the optical density (OD) values for each group were measured via a microplate reader. ③Cell ATP release assay: Transfected cells were harvested and centrifuged. The supernatant was collected, mixed with ATP detection working solution, and then analyzed for fluorescence intensity via a chemiluminescent microplate reader.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/70ab035ab2f10813be859a27.jpeg"},{"id":97142728,"identity":"0d83da61-8fbf-4bee-87a9-0a5552e539a2","added_by":"auto","created_at":"2025-12-01 10:07:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68491,"visible":true,"origin":"","legend":"\u003cp\u003eTransfection efficiency was determined by flow cytometry for different electroporation buffers under identical transfection parameters(n=3). As shown by Tukey's test of one-way analysis of variance, the values in the 1SM group were significantly greater than those in the 2 M group (p\u0026lt;0.01), BTX group (p\u0026lt;0.05), and DMEM group (p\u0026lt;0.05), whereas the values in the 1S and 2S groups were significantly greater than those in the 2 M group (p\u0026lt;0.05). Among these buffers, the 1SM buffer environment yielded the highest transfection efficiency, followed by the 2S and 1S buffers, with no significant differences observed among them (ns).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/c503c2b63d0c09dd6c27d274.jpeg"},{"id":97134052,"identity":"53231327-12d1-4aef-94bf-da756a3bba8d","added_by":"auto","created_at":"2025-12-01 09:13:51","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":166814,"visible":true,"origin":"","legend":"\u003cp\u003eATP release curves of 293T cells with different transfection parameters according to single-factor analysis(n=3). (A) Electric field strength-ATP release curve. Under constant pulse frequency and duration, gradually increasing the electric field strength from low to high increased the degree of cell membrane opening and ATP release. ATP release reached its maximum plateau when the electric field strength increased from 2.0 kV/cm to 2.5 kV/cm. (B) Pulse duration-ATP release curve. With a constant pulse electric field and frequency, progressively longer pulse durations prolonged the open state of the cell membrane channels, leading to increased ATP release. ATP release plateaued at pulse durations of 500 μs or longer. (C) Pulse frequency‒ATP release curve. Under a constant pulse electric field and duration, stepwise increases in pulse frequency increased the number of open channels in the cell membrane, resulting in increased ATP release. The maximum ATP release was achieved when the pulse frequency reached 7 pulses or more.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/a394b7ee35b017523368c288.jpeg"},{"id":97134053,"identity":"466c8930-55ac-471a-a30b-c9f38de7ca06","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":138168,"visible":true,"origin":"","legend":"\u003cp\u003eDetection results of partial groups in the orthogonal experiment. A: Flow cytometry analysis of orthogonal group 6 revealed an EGFP expression rate of 50.2% (The results of the other two independent replicates were respectively 49.2% and 52.0%). B: Flow cytometry analysis of orthogonal group 9 revealed an EGFP expression rate of 78.1% (The results of the other two independent replicates were respectively 79.6% and 77.8%).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/946658374d22dec8a2ea8b62.jpeg"},{"id":97142468,"identity":"5b34b9f6-da81-4ac8-8b69-24cb3c5d76df","added_by":"auto","created_at":"2025-12-01 10:07:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61324,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of expression results for each group in orthogonal design (n=3). The expression for each group was determined from triplicate measurements, with the mean values representing the final results. Tukey's test revealed that Group 1 was significantly different from all the other groups (Groups 2-9) (all p \u0026lt; 0.01). Among the remaining groups, Groups 3 and 9 presented the highest expression levels, which were significantly different from those of most other groups(all p \u0026lt; 0.01). No significant differences were detected between Groups 2, 4, 5, and 7(ns) or between Groups 6 and 8(ns).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/0269a83d52af10f8b3881f55.jpeg"},{"id":97142753,"identity":"d58d6fd3-adff-4cc1-bf18-766ae240b67f","added_by":"auto","created_at":"2025-12-01 10:07:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94099,"visible":true,"origin":"","legend":"\u003cp\u003eOrthogonal nine-group viability analysis of 293T cells (n=3). Group 10 represented the blank control group, with a viability value of 1.00. Groups 1–9 corresponded to orthogonal groups 1–9. A one-way ANOVA with Dunnett's test in demonstrated that, compared to the blank control group, the cell viability of Groups 3, 5, 7, and 9 was extremely significantly decreased (all p \u0026lt; 0.0001,****), while that of Groups 1 and 2 was significantly decreased (p \u0026lt; 0.05,*). No significant differences were detected for Groups 4, 6, and 8 compared to the control group (ns).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/9c1ef001c7c02ff85ac3a3d6.jpeg"},{"id":97134060,"identity":"e20f7ec4-2928-4fb8-b5b8-79dd6473cbde","added_by":"auto","created_at":"2025-12-01 09:13:52","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":309813,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the transfection efficiency, cell viability, and electroporation scores of each orthogonal group of 293T cells (n=3). Among them, group 6 had the highest cell electroporation score; the other groups, in order of decreasing scores, were groups 8, 3, 9, 2, 7, 4, 5, and 1.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/101b966deb0fef30a7cf4c7e.jpeg"},{"id":97142991,"identity":"6e32d680-f2c1-4042-88ef-6aab21d90780","added_by":"auto","created_at":"2025-12-01 10:08:10","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":176643,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of cell viability and transfection efficiency in the validation group. A: A CCK-8 assay was used to assess subsequent cell viability in the validation group, which yielded an average viability of 78.9% (with individual well viability values of 78.2%, 74.5%, and 84.2%). B: Flow cytometry analysis of three independent replicates revealed transfection efficiencies of 82.2%, 77.7%, and 76.3% (only one replicate was shown in the figure).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/d24e9f1fe12d5f22a125c053.jpeg"},{"id":97142459,"identity":"d4020d4e-2154-4e80-96ab-91cbb24fd1f9","added_by":"auto","created_at":"2025-12-01 10:07:37","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":833119,"visible":true,"origin":"","legend":"\u003cp\u003eBrightfield (A) and fluorescence (B) images of positive cells sorted by a sorting-capable flow cytometer. Transfected cells were sorted via flow cytometry and subsequently cultured under standard conditions. Continuous observation over 30 days demonstrated that transfection-positive cells proliferated and passaged normally, with stable fluorescence signals maintained throughout.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/7a3e4e313490268657b04af6.jpeg"},{"id":97248734,"identity":"2ccc0e11-1d37-48cd-af71-f9c9f180eadb","added_by":"auto","created_at":"2025-12-02 13:06:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3397628,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/f5b98d62-5a8c-4c76-82d8-50a559490450.pdf"},{"id":97142028,"identity":"9a48f460-2645-4bd5-8403-d1a8a9e99877","added_by":"auto","created_at":"2025-12-01 10:07:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":877778,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8152999/v1/55a2ae589e4673ba79109daf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Systematic Optimization of 293T Cell Electroporation: Balancing High-Efficiency Gene Delivery with Cell Viability","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdvances in gene engineering and cell biology have underscored the importance of efficient and stable transfection technology for investigating gene function, protein expression, and cell signaling pathways. Electroporation offers a distinct advantage because of its wide applicability, high efficiency, and safety [1]. It is a physical method that achieves gene transfer by creating transient pores in cell membranes through electrical pulses. This physical method is suitable for a variety of cell types, particularly challenging ones such as primary cells, stem cells, and immune cells, which are difficult to transfect via conventional methods [2]. Electroporation is capable of delivering various molecules (DNA, RNA, miRNA, protein, etc.) effectively, with precise control over parameters and reproducible outcomes. Moreover, it circumvents the cytotoxicity associated with chemical reagents, the potential for insertional mutagenesis from viral vectors, and complex biosafety concerns. Consequently, electroporation has emerged as a robust and dependable technology pivotal for cell therapy, gene editing, and fundamental research [3].\u003c/p\u003e\n\u003cp\u003eThe 293T cell line is widely used in gene expression research and virus packaging because of its immortalization ability and rapid proliferation [4].\u003c/p\u003e\n\u003cp\u003eThe transfection efficiency of 293T cells can be significantly impacted by multiple factors, including the transfection buffer, parameters associated with transfection, and the cellular state. The current transfection efficiency of 293T cells is around 60%, which still has a significant gap compared to viral transfection. Therefore,\u0026nbsp;systematically optimizing the electroporation protocol is essential for enhancing the experimental efficiency and reproducibility.\u003c/p\u003e\n\u003cp\u003eIn recent years, existing electroporation devices (such as the BTX ECM830 [5]) have gained widespread application in the field of cell transfection due to their ability to precisely control electroporation-related parameters. These can significantly improve cell transfection efficiency and survival rate by precisely controlling multiple parameters such as the electric field intensity, pulse duration, and pulse number.\u003c/p\u003e\n\u003cp\u003eThis study established an optimized transfection system for these cells on the basis of an orthogonal experimental design, integrating\u0026nbsp;ATP detection, CCK-8 detection, and flow cytometer detection techniques:\u003c/p\u003e\n\u003cp\u003e1. Cellular ATP release assay: This assay quantifies the extracellular ATP concentration following electroporation, directly indicating the effects of various transfection parameters on transient membrane permeability [6](extent of pore opening), serving as a dynamic metric for parameter evaluation;\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp;CCK-8 assay for cell viability analysis: Cells were co-incubated with the CCK-8 reagent for 1–2 hours following electroporation (24 hours post-transfection). The absorbance at 450 nm was measured via an ELISA reader to evaluate long-term survival rates and determine the damage threshold of the transfection parameters on the cell proliferation capacity.\u003c/p\u003e\n\u003cp\u003e3. Flow cytometry is employed to measure transfection efficiency accurately by assessing the expression levels of fluorescently labeled plasmids or reporter genes, such as GFP, in transfected cells. In this study, the transfection plasmid, derived from the GV712 plasmid, contains the EGFP gene GV712-EGFP, which is approximately 7.5 kP in length, enabling the exclusion of interference from cell debris or nonspecific signals.\u003c/p\u003e\n\u003cp\u003eFurthermore, this study utilizes the \"Electroporation score\" (EPS) [7] as a comprehensive assessment tool. The EPS calculation formula combines cell viability data from CCK-8 assays with transfection efficiency data from flow cytometry, the EPS effectively represents the balance between \"efficiency-viability\" for various parameter combinations. This approach overcomes the constraints associated with optimizing a singular metric.\u003c/p\u003e\n\u003cp\u003eThis study utilized a multi-index linkage strategy to determine the optimal electroporation parameters (electric field voltage, pulse duration, and pulse number) for transfecting 293T cells via a BTX transfection instrument. This research not only identified the ideal parameter window but also elucidated the nonlinear correlation between cell membrane permeability (ATP release) and long-term activity (CCK-8 OD value). The established protocol offers a standardized approach for optimizing electroporation parameters for 293T cells. The modular design of the protocol, which includes single-factor analysis, orthogonal screening, CCK-8/flow detection, and EPS quantitative comparison, can be extrapolated to gene delivery studies involving challenging cell lines. This methodology provides a solid foundation for ensuring experimental reproducibility in gene editing, virus packaging, and related research fields.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003e293T cells(ATCC; VA; USA), DMEM (biosharp; Cat. No. BL305A; Lot. No.00625678CJ; China), BTX buffer (BTXpress; CatalogNo.47\u0026thinsp;\u0026minus;\u0026thinsp;0002; Lot:4000079306, USA), Chica buffers(1S, 2S,1M, 2M, 1SM; For specific formulations, please refer to Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in the supplementary materials), Electroporation cuvette (BTX, USA), ATP chemiluminescence assay kit (Servicebio; Cat:G4309-48T; Lot:MPC2409061; China), Cell Counting Kit-8 (biosharp; Cat. No. BS350B; Lot: 23360953; China), PBS (1\u0026times;) (SparkJade; REF: CR0014-500ML; LOT: FFELY; China), Fetal bovine serum standard (Cell max; Cat No. SA301.02. V;lot No.20221220; China), 0.25% Trypsin-EDTA(1\u0026times;)(GOONIE༛Cat: G100-502༛Lot༚250322; China), Penicillin/Streptomycin(100\u0026times;)(Goonie; Cat༚100\u0026ndash;503; Lot༚LGM241204; China), Flow Cytometer(FACSVerse; BD; USA), Synergy Neo2 Microplate Reader(BioTek; USA༉, ECM830 Square Wave Electrical Converter(BTX; USA), Sorting Flow Cytometer (FACSAria III; BD; USA), GV712-EGFP Plasmid(GENECHEN; China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Ethics Approval\u003c/h2\u003e\u003cp\u003eThe 293T cell line used in this study was purchased from the American Type Culture Collection (ATCC) through official laboratory channels, and the source was compliant. This cell line is a commercial immortalized cell line. According to the current ethical standards at home and abroad, such research is usually exempt from ethical review. This study is an in vitro cell experiment that does not involve human embryos or clinical applications and meets the ethical requirements of biomedical research.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Plasmids and Cloning\u003c/h2\u003e\u003cp\u003eThe GV712-EGFP plasmid used in this experiment was synthesized and provided by Jikai Gene Co., Ltd. Using the GV712 plasmid as the design vector, the plasmid was cut after the CMV promoter protein gene with XbaI enzyme, and then a gene fragment containing Glycosylation sequence protein GNSTM with-3-EGFP-3FLAG was inserted to construct (-CMV-GNSTM-3-EGFP-3FLAG-puromycin-).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Cell culture\u003c/h2\u003e\u003cp\u003e293T cells were cultured in complete DMEM (containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin double antibody) in a constant-temperature incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUpon reaching the logarithmic growth phase (approximately 80\u0026ndash;90% confluence), the old culture medium was aspirated, and the cell monolayer was gently rinsed with phosphate-buffered saline (PBS). An appropriate volume of trypsin solution was then applied to dissociate the adherent cells. After complete digestion, the trypsin activity was neutralized by adding complete medium, and a single-cell suspension was obtained by gentle trituration. The cell suspension was subsequently centrifuged to form a pellet, followed by cell counting. The cells were then either passaged at a suitable ratio or subjected to a second centrifugation and resuspended in electroporation buffer at an optimal density for subsequent electroporation experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Electroporation and optimization scheme\u003c/h2\u003e\u003cp\u003eUsing a single-factor design to control variables, a specific high-efficiency buffer with the highest transfection efficiency and best protective effect for 293T cells was screened from seven electroporation buffers. Subsequently, the maximum tolerable ranges for pulse voltage, pulse duration, and pulse number in 293T cells were sequentially determined using the ATP assay.\u003c/p\u003e\u003cp\u003eUnder the selected specific buffer conditions, appropriate levels were chosen from the tolerance ranges of the three parameters for subsequent orthogonal design experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003e(1) Single-factor design\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003e1) Selection of cell-specific high-efficiency buffer\u003c/h3\u003e\n\u003cp\u003e293T cells at the logarithmic growth phase (approximately 80% confluence) were harvested via trypsinization, collected by centrifugation, and counted via a hemocytometer. The cells were resuspended in the respective buffers\u0026mdash;DMEM, BTX buffer, and Chica buffers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] (including 1 SM, 1 S, 2 S, 1 M, and 2 M)\u0026mdash;at a density of 5\u0026times;10⁶ cells/mL. The corresponding amount of GV712-EGFP plasmid (5 \u0026micro;g per 400 \u0026micro;L of cell suspension) was added to each group. A volume of 400 \u0026micro;L of the mixture was gently transferred into a 2 mm electroporation cuvette, and the cuvette was tapped gently to eliminate air bubbles. Transfection was performed via identical parameters for all groups, with each condition tested in triplicate. The data are presented as the means of three replicates. To compare the transfection efficiency across the different electroporation buffers, one-way analysis of variance (ANOVA) was conducted. If significant intergroup differences were detected, Tukey test was applied for multiple comparisons. All the statistical analyses were performed via GraphPad Prism (version 9.5.0) software.\u003c/p\u003e\n\u003ch3\u003e2) Optimization of electroporation parameters\u003c/h3\u003e\n\u003cp\u003eThe extent of cell membrane permeabilization under a given set of electroporation parameters was determined by measuring the amount of adenosine triphosphate (ATP) released extracellularly immediately following electric pulse delivery, according to the ATP content assay described by Marie-Pierre et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This assay indirectly reflects the degree of pore formation in the plasma membrane by quantifying the efflux of intracellular ATP molecules, thereby helping to establish tolerable ranges for key parameters: voltage, pulse duration, and pulse number.\u003c/p\u003e\u003cp\u003eElectric field strength is the critical determinant of successful membrane permeabilization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Owing to the fluid nature of the phospholipid bilayer, an electric field strength below a specific critical threshold (CT) fails to effectively disrupt membrane integrity, preventing significant ATP release. Conversely, when the field strength exceeds a certain highest threshold (HT), excessive energy causes irreparable damage to the phospholipid bilayer, leading to cell rupture and death\u0026mdash;a process known as irreversible electroporation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePulse duration and pulse number are essential parameters for sustaining membrane pores. Once pores are successfully created by an applied electric field, the inherent properties of the membrane drive rapid resealing. An appropriate pulse duration and number help maintain the existence and quantity of pores in the phospholipid bilayer without compromising cell viability, thereby facilitating the release of intracellular ATP molecules.\u003c/p\u003e\u003cp\u003eOn the basis of the aforementioned principles, a controlled variable approach was employed. Under constant conditions, experiments were designed with gradient increases in the transfection voltage, pulse duration, and pulse frequency. The specific experimental design is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Following electroporation, the cells were removed under appropriate centrifugation conditions, and the supernatant was collected. Detection was performed via a chemiluminescent ATP assay kit, and the corresponding ATP release curves were plotted.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSingle-Factor Analysis Design\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFactor Tested\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFixed Conditions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBuffer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAll other transfection parameters held constant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVoltage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePulse duration: 100 \u0026micro;s, number of pulses: 3, and all other transfection parameters held constant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePulse Duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVoltage: 300V, number of pulses: 3, and all other transfection parameters held constant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of Pulses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVoltage: 300V, pulse duration: 100 \u0026micro;s, and all other transfection parameters held constant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e(2) Orthogonal design\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eOn the basis of the three ATP content detection curves plotted from the single-factor analysis, three suitable parameter levels were selected, and a three-factor, three-level orthogonal experimental design (L9 (3\u0026sup3;)) was conducted, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results of the orthogonal experiments were compared through range analysis and analysis of variance to determine the optimal combination of the three transfection parameters for validation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1: Orthogonal design\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLevel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eFactor\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA: Voltage (V)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB: Duration (\u0026micro;s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC: Number of Pulses\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e2: Orthogonal design experiment groups\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Calculation of Electroporation Scores\u003c/h2\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Assessment of cell viability via the CCK-8 assay\u003c/h2\u003e\u003cp\u003eFollowing an orthogonal design, the cells were electrotransfected via the ECM830 square wave electrical converter. After electroporation, the electrode cups were allowed to stand at room temperature for 10\u0026ndash;15 minutes. The cells were then slowly resuspended via a dedicated Pasteur pipette to ensure uniform dispersion. A suspension of 2 \u0026times; 10⁴ cells from each group was seeded into 96-well plates containing fresh medium prewarmed to 37\u0026deg;C in a water bath, with three parallel wells per group. After all groups were seeded, the plates were incubated at 37\u0026deg;C for 24 hours. Following the instructions of the CCK-8 assay kit, the absorbance at 450 nm was measured for each transfected cell group via a Synergy Neo2 microplate reader. The specific workflow is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The data were processed via GraphPad Prism 9.5.0 software. Cell viability was calculated as follows: Cell viability(%) = (OD transfection experimental \u0026ndash; OD control medium group)/(OD blank cell \u0026ndash; OD control medium group)\u0026times;100%. Dunnett's test in one-way ANOVA was performed between each group and the blank cell group to assess statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Flow cytometry detection of EGFP fluorescent signal\u003c/h2\u003e\u003cp\u003eThe uniformly resuspended electroporated cells were transferred by group into six-well plates containing fresh medium prewarmed to 37\u0026deg;C. After 24 hours of culture, the cell status was observed, and a half-medium change was performed. The cells were further cultured for approximately 72 hours, when they had largely recovered and reached an appropriate density. The cells were then trypsinized, collected, and resuspended in PBS (300 \u0026micro;l) to prepare single-cell suspensions. Transfection efficiency was assessed via a flow cytometer. Data analysis was performed via Flow Jo software (version 10.8.1). Statistical analysis was conducted via one-way ANOVA followed by Tukey test via GraphPad Prism software (version 9.5.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Calculation of electroporation score\u003c/h2\u003e\u003cp\u003eTo synthesize the effects of transfection parameters, the Electroporation Score (EPS) concept proposed by Chicaybam L [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] in 2017 was specifically applied. EPS was calculated as: EPS\u0026thinsp;=\u0026thinsp;Cell Viability (%) \u0026times; Expression (%) / F, where the fitting factor F was 50 for adherent cell lines and 100 for non-adherent cell lines. This adjustment facilitated graphical representation and provided a more comprehensive and intuitive quantification of transfection outcomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Determination of an efficient buffer\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, under identical electroporation conditions, different buffers exhibited significantly different transfection efficiencies. Among them, buffers 1 SM, 2 S, and 1 S demonstrated superior transfection efficiency compared with other buffers, with 1 SM buffer achieving the highest transfection efficiency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Cellular ATP release curve\u003c/h2\u003e\u003cp\u003eOn the basis of the single-factor analysis principle described in Section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.2.2\u003c/span\u003e, with a fixed pulse duration of 100 \u0026micro;s and three pulses administered, the pulse voltage was set in a gradient. Following electroporation, the cell suspensions were collected from each group of electroporation cuvettes and centrifuged at 1000 rpm for 3 minutes, and the supernatants from each group were obtained. The supernatants were transferred into white opaque 96-well plates (with three replicate wells per group). The prepared ATP detection working solution was mixed with the supernatants according to the protocol of the ATP detection kit (Sevier). Luminescence values were then rapidly measured for each well via a chemiluminescence-detecting Synergy Neo2 microplate reader.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A, when the electric field strength exceeded 0.25 kV/cm (corresponding to an electroporation voltage greater than 50 V), the electric shock successfully punctured the cell membrane, and intracellular ATP began to be released into the external environment. This electric field strength represents the critical threshold (CT) for cell membrane opening. As the electric field strength continued to increase, the extent of membrane opening also increased. When the electric field strength increased from 2.0 kV/cm to 2.5 kV/cm, the amount of ATP released by the cells reached a maximum plateau. Observation of the cells after transfection revealed that the electric field intensity at this point caused irreversible damage to the cell membrane, ultimately leading to cell rupture and death. Therefore, 2.5 kV/cm was determined to be the highest threshold (HT) for membrane opening that the cells could withstand. On the basis of these findings, the electric field strength levels selected for the orthogonal design were 1.5 kV/cm, 1.75 kV/cm, and 2.0 kV/cm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFollowing the same protocol, a fixed electric field strength of 1.5 kV/cm was selected from the range of tolerable electric field strengths determined experimentally. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A, the voltage applied at this point effectively opened the cell membrane while maintaining cellular viability with minimal impact. The pulse count was held constant at 3 pulses, while the pulse duration was increased in a gradient manner. The ATP release levels were then measured across each group to determine the range of pulse durations the cells could tolerate, which provided an appropriate design range for subsequent orthogonal experiments. The final results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-B. On the basis of these experimental results, the pulse duration levels selected for the orthogonal design were 100 \u0026micro;s, 300 \u0026micro;s, and 500 \u0026micro;s.\u003c/p\u003e\u003cp\u003eThe experimental method for pulse frequency was identical to the two aforementioned detection methods, with the final results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-C. After comprehensive consideration of the orthogonal design, the frequency levels were ultimately selected as 2, 4, and 6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Orthogonal Design\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Selection of electric field intensity, pulse time, and pulse number gradient\u003c/h2\u003e\u003cp\u003eOn the basis of the optimal factor levels identified through single-factor analysis, the experiment was designed according to the orthogonal design table with three factors at three levels (L9 (3\u0026sup3;)). The detailed experimental parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Transfection efficiency detection\u003c/h2\u003e\u003cp\u003eFollowing the method described in Section \u003cspan refid=\"Sec13\" class=\"InternalRef\"\u003e2.3.2\u003c/span\u003e, cells that had recovered from the confluent state after transfection were digested and harvested. After resuspension in PBS buffer, the transfection efficiency of cells in each orthogonal group was measured via a flow cytometer (BD FACS Verse). Each group was analyzed in triplicate, and the average value was recorded. The flow cytometry results for the selected groups in the orthogonal experiments are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The transfection efficiency results for all the experimental groups are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3 Cell viability in each group\u003c/h2\u003e\u003cp\u003eFollowing the assay method described in Section \u003cspan refid=\"Sec16\" class=\"InternalRef\"\u003e3.1\u003c/span\u003e, cells from each group subjected to electroporation were seeded into 96-well plates for culture, with three parallel wells per group. Cell viability was assessed via a CCK-8 assay. The mean viability across the three wells per group served as the final viability result for that group. The viability of the blank group was set as 1.00. All other groups were normalized to the blank group for comparison, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eCompared with the blank group, Groups 4, 6 and 8 did not significantly differ in viability across groups(ns), indicating that the transfection parameters used in Group 6 did not cause irreversible substantial damage to the cells themselves. For the transfection parameters of the other groups, the cells were affected to varying degrees, particularly in Group 9, where cell viability was significantly impacted (****) by the corresponding parameters.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.4 Electroporation scoring and orthogonal experimental results validation\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe transfection efficiencies and cell viabilities of the nine orthogonal cell groups, obtained via flow cytometry and CCK-8 assays, respectively, were determined. The electroporation scores for each group were calculated via the formula outlined in Section \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e2.3.3\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1).\u003c/p\u003e\u003cp\u003eOn the basis of the electroporation scores, the range method and variance method from the orthogonal experiments were applied to analyze and determine the optimal levels of the three factors within the orthogonal design.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026thinsp;\u0026minus;\u0026thinsp;1: Orthogonal design experiment results\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eExpression(%)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eViability(%)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eEPS(%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e85.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e12.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e28.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e84.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e49.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e63.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e55.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e70.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e92.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e44.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e34.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e53.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e36.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e50.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e93.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e94.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e33.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e70.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e47.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e91.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e87.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e78.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e57.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026thinsp;\u0026minus;\u0026thinsp;2: Range analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRange Analysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44.160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33.813\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e64.823\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e58.443\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e57.577\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e50.263\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63.990\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e74.203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e51.507\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e19.830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.390\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.560\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e3: Analysis of Variance\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnalysis of Variance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSum of Squares (SS)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDegrees of Freedom (df)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e628.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.560\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2346.187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.092\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e390.873\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.348\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eError\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3365.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThrough analysis of the range (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;2) and variance (Table\u0026nbsp;\u0026lt;link rid=\"tb6\"\u0026gt;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u0026lt;/link\u0026gt;\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e) of the orthogonal design experiment results, the levels with the highest K values among the three factors A, B, and C were A3, B3, and C1, respectively. Thus, the optimal theoretical combination for 293T cells within this orthogonal range was A3B3C1, corresponding to transfection parameters of 2.0 kV/cm, 500 \u0026micro;s, and 2 pulses. Among these factors, factor B presented the highest R value, indicating that pulse duration was the most influential factor affecting cell transfection in this orthogonal design. The analysis of variance further corroborated this conclusion.\u003c/p\u003e\u003cp\u003eAccording to the results of the orthogonal design experiment, a validation group was designed using the following transfection parameters: 400 V, 500 \u0026micro;s, and 2 pulses, with all other factors held constant for the transfection of 293T cells. The final results of three replicate experiments were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. While ensuring that cell viability was no lower than 70.0%, the transfection efficiency reached (78.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1) %, with a score of 124.3. This score exceeded those of all orthogonal groups. Compared with the groups shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the validation group successfully enhanced the transfection efficiency while maintaining cell viability. The results met expectations, confirming the validity of this orthogonal design.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Positive Cell Sorting\u003c/h2\u003e\u003cp\u003eThe 293T cells were transfected using the optimized transfection parameters obtained through orthogonal design and a multi-index combined system. After reaching a stable state, the cells were digested, collected, and resuspended in complete medium to form a single-cell suspension at a concentration of 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml. Using a sorting flow cytometer, positive cells expressing EGFP protein were sorted under the 488 nm excitation channel, ultimately yielding highly positive cells (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). After 30 days of continuous culture, the sorted cell population maintained stable growth and exhibited sustained, uniform EGFP fluorescence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Combinations of Multiple Methods\u003c/h2\u003e\u003cp\u003eThis study adopted an orthogonal design as the research approach, integrating multiple indicator methods such as the ATP assay and the CCK-8 assay to establish an efficient and systematic electroporation condition optimization system. This approach provides a reliable solution for rapidly screening optimal parameter combinations that balance transfection efficiency and cell viability, effectively overcoming the challenges inherent in traditional electroporation condition optimization. These challenges include reliance on single qualitative indicators, low transfection efficiency, high experimental blindness, and difficulty in simultaneously addressing membrane permeability and cell viability.\u003c/p\u003e\u003cp\u003eOrthogonal design represents a highly efficient multifactor optimization strategy widely applied in fields process, formulation, or parameter optimization [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Its core advantage lies in scientifically and efficiently analyzing and determining optimal combinations within multifactor, multilevel problems with minimal experimental runs. These strengths perfectly align with the objectives of optimizing electroporation experiments for cells.\u003c/p\u003e\u003cp\u003eAmong the numerous factors involved in cell electroporation experiments, the three most critical factors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] are: (1) electric field, whether it can successfully \"open\" the cell membrane without compromising subsequent cell viability. A low electric field failed to effectively puncture the cell membrane, whereas a high electric field caused irreparable damage to the cell membrane [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]; (2) time and number of pulses, short durations or few pulses failed to sustain the \"channel\" opened by the electric field, whereas prolonged durations or excessive pulses resulted in prolonged membrane opening and excessive shock, leading to cell death.\u003c/p\u003e\u003cp\u003eHowever, since different cell lines exhibit varying tolerances to electroporation, confirming the key transfection factors and rationally selecting their parameter levels are crucial in orthogonal design. The ATP assay served as a suitable solution. The amount of ATP released by electroporated cells reflects the extent of membrane opening. This method allowed for the gradual determination of tolerable ranges for the three factors, providing objective and reasonable basis for selecting the level gradients of each factor.\u003c/p\u003e\u003cp\u003eThe use of the CCK-8 assay to assess cell viability post transfection not only quantified cellular recovery following electroporation but also, when analyzed alongside ATP detection curves, revealed the nonlinear relationship between membrane permeability and long-term viability. Specifically, increased membrane permeability was correlated with greater ATP release, whereas conversely, cellular activity decreased.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Electroporation score\u003c/h2\u003e\u003cp\u003eThe evaluation of a transfection parameter's effect on electroporated cells involves two main aspects: the expression rate of transfected cells and their subsequent viability [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The primary objective of this study was to identify the optimal overall performance of the transfection parameters through various detection methods rather than pursuing the highest expression rate or highest survival rate in isolation. Therefore, to avoid the accuracy of the final results being affected by an extreme positive or negative value of a single parameter, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, when considering only the cell expression rate, the expression rate of orthogonal group 9 reached 78.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which was higher than that of the other experimental groups and the control group. However, the cell viability in this group was significantly affected by the transfection parameters, with cell viability less than 40% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Therefore, a comprehensive evaluation integrating both aspects was essential to achieve the ultimate objective of this study.\u003c/p\u003e\u003cp\u003eThe EPS serves as a comprehensive quantitative metric capable of rationally converting two or more evaluation indicators. Through computational methods (as outlined in Section \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e2.3.3\u003c/span\u003e), a single score was generated to holistically assess the \"quality\" of an electroporation experiment. Through simple mathematical operations, it integrates transfection efficiency and cell viability\u0026mdash;two metrics that often constrain each other\u0026mdash;transforming complex multi objective trade-offs into an intuitive composite score. This approach enabled clear quantification and comparison of the equilibrium between transfection efficiency and cell viability under different parameter conditions. Subsequent analyses could then precisely determine the extent to which specific transfection parameters impact the overall cellular state.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Analysis of the orthogonal design experiment results\u003c/h2\u003e\u003cp\u003eDrawing on the range and variance analysis of the orthogonal design experiment results (Tables\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;2 and 3\u0026ndash;3), within the designed orthogonal range, pulse duration (factor B) was the primary factor influencing EPS (R\u0026thinsp;=\u0026thinsp;38.390, F\u0026thinsp;=\u0026thinsp;2.092), with its significance far exceeding that of electric field voltage (A) and pulse frequency (C). This finding provides crucial insight: pulse duration directly determines the duration of cell transfection, thereby influencing the influx of exogenous substances and the self-repair process of the cell membrane. Insufficient duration resulted in inadequate delivery of the target plasmid, whereas excessive duration caused irreversible membrane damage and imbalances in ions and other substances, ultimately leading to cell death. The optimal parameter combination A3B3C1 (400 V, 500 \u0026micro;s, 2 pulses) achieved a balance between high transfection efficiency and high cell viability precisely because it identified the optimal equilibrium between the membrane permeability window and cellular tolerance. This further demonstrated the comprehensiveness and rationality of using EPS as an orthogonal design experiment result evaluation metrics. It also demonstrated the rationality of this optimization system that combines orthogonal design with multiple indicators.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Acquisition of Fully Positive Cells\u003c/h2\u003e\u003cp\u003eHighly efficient and stable transfection meant that more cells successfully incorporated and expressed the target gene (EGFP), thereby forming a high proportion of positive cell populations within the cell population. This advantage ensured that when using a sorting-type flow cytometer for positive cell sorting in subsequent steps, there was an adequate number of target cells with clear signals, significantly simplifying the sorting process, reducing sorting time, and lowering the experimental time and resource costs. Therefore, this optimized scheme not only improved the transfection efficiency itself but also provided technical support for the subsequent efficient acquisition of fully positive cell clones, demonstrating the significant gain in overall experimental workflow efficiency through systematic optimization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Research limitations\u003c/h2\u003e\u003cp\u003eThis study has several limitations, too. First, all the optimization work was conducted using the 293T cell line. Although this approach has shown theoretical potential for extension to other difficult-to-transfect cell types, further validation is needed. Second, the study indirectly assessed membrane permeability through ATP release but did not investigate the dynamic mechanisms of post electroporation membrane repair or cell death pathways. As noted by Kotnik et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the mechanisms of membrane resealing after electroporation and their relationship with cell survival remain significant challenges in the field. Future studies can employ molecular dynamics simulations combined with real-time imaging techniques to further elucidate these dynamic processes. Additionally, the optimal parameters identified in this protocol were specific to a particular electroporation device (ECM830; BTX; USA), and their applicability to other instruments might be limited owing to differences in electric field design. Future studies can verify this scheme in more cell models and further deepen the applicability depth and reliability of this optimized system by combining membrane repair mechanism research with functional endpoint indicators.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study systematically optimized key parameters for electroporation transfection in 293T cells and established an efficient screening system for transfection conditions on the basis of an orthogonal experimental design and multi-indicator integration. ATP release assays define the dynamic range of changes in cell membrane permeability. The CCK-8 assay and flow cytometry were employed to evaluate long-term survival rates and transfection efficiency in transfected cells, respectively. The introduction of EPS serves as a comprehensive metric that effectively balances the trade-off between transfection efficiency and cell viability.\u003c/p\u003e\u003cp\u003eThe experimental results demonstrated that under optimized electroporation buffer (1 SM buffer) and the theoretically optimal parameter combination (400 V, 500 \u0026micro;s, 2 pulses), both the transfection efficiency and cell viability in 293T cells were significantly enhanced, resulting in the highest EPS score. This finding validates the effectiveness and reliability of the orthogonal design and multi-indicator integration strategy. Furthermore, it was also significantly improved the efficiency of subsequent flow cytometric sorting to obtain fully positive cells, making the process more convenient and efficient, and greatly saving time and experimental costs.\u003c/p\u003e\u003cp\u003eThis study provides not only a standardized, reproducible protocol for electroporation of 293T cells but also a modular optimization strategy that can be extended to other cell types. It offers methodological support and a reliable optimization plan for research in gene editing and cell therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: M.S.Z, B.Y, Z.Y.Z. Data curation and Formal analysis: M.S.Z, M.R.L, Q.T.Z, B.Y. and Z.Y.Z. ATP detection: M.S.Z, M.R.L, D.Y.Z, Q.Y.Y. and Z.Z.Y. Flow Cytometry Analysis: M.S.Z, M.R.L, Z.Y.Z. Funding acquisition: B.Y. Resources: M.S.Z, B.Y, Z.Y.Z. Supervision: Z.B.G, J.X.W, N.P.Z and F.F.C. Writing—original draft: M.S.Z. Writing—review \u0026amp; editing: Q.T.Z, B.Y, Z.Y.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Department of Traditional Chinese Pharmacy, School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. \u003csup\u003e2\u003c/sup\u003e Department of Pharmacology, Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. \u003csup\u003e3\u003c/sup\u003e Department of Traditional Chinese Pharmacy, Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. \u003csup\u003e4\u003c/sup\u003e Department of Neurosurgery, Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. \u003csup\u003e5\u003c/sup\u003e Department of Traditional Chinese Pharmacy, School of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, Shandong. \u003csup\u003e6\u003c/sup\u003e Department of Medical Immunology, School of Clinical and Basic Medicine, Shandong First Medical University \u0026amp; Shandong Academy of Medical Sciences, Jinan, Shandong.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun, H., Yu, L., Chen, Y., Yang, H. \u0026amp; Sun, L. Analysis of In Situ Electroporation Utilizing Induced Electric Field at a Wireless Janus Microelectrode. \u003cem\u003eMicromachines (Basel)\u003c/em\u003e. \u003cb\u003e15\u003c/b\u003e (7), 819 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSioud, M. Optimized siRNA Delivery into Primary Immune Cells Using Electroporation. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e \u003cb\u003e2115\u003c/b\u003e, 119\u0026ndash;131 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, F. et al. Advanced micro/nano-electroporation for gene therapy: recent advances and future outlook. \u003cem\u003eNanoscale\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (22), 10500\u0026ndash;10521 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, C. et al. Engineering a HEK-293T exosome-based delivery platform for efficient tumor-targeting chemotherapy/internal irradiation combination therapy. \u003cem\u003eJ. Nanobiotechnol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (1), 247 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcIntyre, D. A. \u0026amp; Harlander, S. K. Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e (3), 604\u0026ndash;610 (1989).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGibot, L. et al. Calcium Delivery by Electroporation Induces In Vitro Cell Death through Mitochondrial Dysfunction without DNA Damages. \u003cem\u003eCancers (Basel)\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e (2), 425 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChicaybam, L. et al. An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells. \u003cem\u003eFront. Bioeng. Biotechnol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 99 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, X. L. et al. Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e (19), 10195\u0026ndash;10215 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChicaybam, L., Sodre, A. L., Curzio, B. A. \u0026amp; Bonamino, M. H. An efficient low cost method for gene transfer to T lymphocytes. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e (3), e60298 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRols, M. P. \u0026amp; Teissi\u0026eacute;, J. Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e (5), 1089\u0026ndash;1098 (1990).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVanderBurgh, J. A., Corso, T. N., Levy, S. L. \u0026amp; Craighead, H. G. Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (1), 6857 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, N. et al. l. Irreversible Electroporation: An Emerging Immunomodulatory Therapy on Solid Tumors. Front Immunol. ;12:811726. (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuna, J., Jaynes, J., Xu, H. \u0026amp; Wong, W. K. Orthogonal array composite designs for drug combination experiments with applications for tuberculosis. \u003cem\u003eStat. Med.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e (17), 3380\u0026ndash;3397 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu, C. et al. Optimizing glycerosome formulations via an orthogonal experimental design to enhance transdermal triptolide delivery. \u003cem\u003eActa Pharm.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e (1), 135\u0026ndash;146 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampillo-Davo, D. et al. The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (3), 396 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampelo, S. N., Huang, P. H., Buie, C. R. \u0026amp; Davalos, R. V. Recent Advancements in Electroporation Technologies: From Bench to Clinic. \u003cem\u003eAnnu. Rev. Biomed. Eng.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 77\u0026ndash;100 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTimmins, L. M. et al. Selecting a Cell Engineering Methodology During Cell Therapy Product Development. \u003cem\u003eCell. Transpl.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 9636897211003022 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKotnik, T., Rems, L., Tarek, M. \u0026amp; Miklavčič, D. Membrane Electroporation and Electropermeabilization: Mechanisms and Models. \u003cem\u003eAnnu. Rev. Biophys.\u003c/em\u003e \u003cb\u003e48\u003c/b\u003e, 63\u0026ndash;91 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Electroporation, ATP assay, CCK-8, Transfection efficiency, Cell viability, Electroporation score","lastPublishedDoi":"10.21203/rs.3.rs-8152999/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8152999/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eStandard 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. \u003cstrong\u003eResults:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion: \u003c/strong\u003eThe 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.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e 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.\u003c/p\u003e","manuscriptTitle":"Systematic Optimization of 293T Cell Electroporation: Balancing High-Efficiency Gene Delivery with Cell Viability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 09:13:47","doi":"10.21203/rs.3.rs-8152999/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-20T15:49:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254174940428880786353813014334504433408","date":"2026-05-07T13:56:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301497341869794807478936037994146303558","date":"2026-04-07T14:16:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-23T11:32:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79594250962016840997721990704981561408","date":"2025-12-03T10:32:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-28T10:36:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-25T05:17:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-20T14:27:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-20T14:24:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-19T08:45:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca58bb35-dd0c-490e-b286-0a11cad756f5","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-20T15:49:05+00:00","index":58,"fulltext":""},{"type":"reviewerAgreed","content":"254174940428880786353813014334504433408","date":"2026-05-07T13:56:20+00:00","index":57,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58829102,"name":"Biological sciences/Biological techniques"},{"id":58829103,"name":"Biological sciences/Biotechnology"},{"id":58829104,"name":"Physical sciences/Engineering"}],"tags":[],"updatedAt":"2025-12-01T09:13:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 09:13:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8152999","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8152999","identity":"rs-8152999","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}