High-efficiency electroporation buffer, preparation method and application thereof

Abstract

This invention discloses a high-efficiency electrotransfection buffer and its preparation and application, belonging to the field of eukaryotic cell gene delivery. Addressing the problems of low electrotransfection efficiency, high cell death rate, impaired differentiation potential, and expensive reagents in existing C2C12 mouse myoblasts, this invention provides a dedicated buffer formulation containing 220 mM sucrose, 30 mM mannitol, 20 mM trehalose, 10 mM HEPES, 1.2 mM MgCl2, and supplemented with 10 μM resveratrol and 0.005% Pluronic F-68, at pH 7.4. During preparation, a basal stock solution and an additive stock solution are prepared separately, mixed, and the pH is adjusted. In use, plasmids or siRNA are mixed with cells in a specific ratio for electrotransfection, followed immediately by resuscitation with pre-warmed culture medium. This invention's buffer achieves a GFP-positive transfection rate of 62.8% while maintaining a high cell viability (OD). 45 =0.46, 24h), and does not affect the subsequent myogenic differentiation ability of C2C12 cells, with a cost of less than one-thousandth of commercial products, making it suitable for high-throughput gene overexpression and knockdown experiments.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering and gene transduction, specifically relating to a high-efficiency electrotransfection buffer, its preparation method, and its application. Background Technology

[0002] The C2C12 mouse myoblast cell line is a widely used in vitro cell model in studies of skeletal muscle development, differentiation, and the mechanisms of muscle-related diseases. These cells proliferate rapidly under serum-sufficient conditions and can fuse to form multinucleated myotubes under low-serum induction, highly mimicking the in vivo skeletal muscle differentiation process. Therefore, efficient and low-toxicity exogenous gene delivery techniques are crucial for conducting gene overexpression, knockdown, and functional regulation experiments in these cells.

[0003] Currently, the most commonly used transfection method in laboratories is liposome-mediated transfection. This technique involves encapsulating DNA or RNA into a complex using cationic liposomes, which then delivers exogenous nucleic acids into the cell via endocytosis. However, liposome transfection has significant drawbacks: firstly, its transfection efficiency is greatly affected by cell type and serum, and is generally low in C2C12 cells; secondly, liposome reagents are expensive and have some cytotoxicity, making them unsuitable for large-volume or high-throughput transfection experiments. To overcome these shortcomings, electroporation (electroporation) technology has emerged. It uses a transient electric field to create reversible micropores in the cell membrane, allowing exogenous substances to directly enter the cytoplasm. This technique offers advantages such as ease of operation, good reproducibility, and no chemical carrier toxicity.

[0004] Despite its significant theoretical advantages, electrotransfection still faces numerous technical bottlenecks in its practical application with C2C12 cells. Currently, there is a lack of dedicated electrotransfection buffer formulations optimized for the membrane characteristics and growth / differentiation features of C2C12 cells. Researchers mostly use commercially available universal kits or universal formulations reported in the literature. However, commercially available kits are expensive, significantly increasing experimental costs; and the published universal formulations generally have low transfection efficiency in C2C12 cells, making it difficult to meet the demands of high-throughput screening.

[0005] More critically, existing electroporation systems generally exhibit a negative correlation between transfection efficiency and cell viability: while increasing the electric field strength or optimizing the procedure can improve nucleic acid delivery efficiency, it often leads to irreversible damage to the cell membrane, significantly increasing mortality. Furthermore, the electroporation process itself may adversely affect the myogenic differentiation potential of C2C12 cells, making it difficult for transfected cells to differentiate normally into myotubes, thus limiting downstream functional studies. Therefore, there is an urgent need in this field for an electroporation buffer specifically designed for C2C12 cells that can balance high transfection efficiency, high cell viability, and maintain differentiation capacity, while also being cost-effective, along with its corresponding usage method. Summary of the Invention

[0006] This invention discloses a high-efficiency electroporation buffer, its preparation method, and its applications. The buffer comprises 220 mM sucrose, 30 mM mannitol, 20 mM trehalose, 10 mM HEPES, 1.2 mM MgCl2, and contains 10 μM resveratrol and 0.005% Pluronic F-68, with a pH of 7.4. This buffer is easy to prepare and, when used with the Lonza 4D electroporator CA133 program, can achieve a GFP-positive transfection rate of 62.8%, while maintaining high cell viability and not affecting myoblastic differentiation ability, at a cost less than one-thousandth that of commercially available products.

[0007] On the one hand, the present invention provides a high-efficiency electroporation buffer solution, which adopts the following technical solution: A high-efficiency electroporation buffer comprising the following components at final concentrations: Sucrose 200~250 mM; Mannitol 20~40 mM; Trehalose 10~30 mM; HEPES 5~15 mM; MgCl2 0.5~2.0 mM; Resveratrol 5~20 μM; Pluronic F-68 0.001%~0.01% (w / v); The pH of the buffer solution is 6.8~7.6.

[0008] Preferably, the component comprises the following final concentrations: 220 mM sucrose; Mannitol 30 mM; Trehalose 20 mM; HEPES 10 mM; MgCl2 1.2 mM; Resveratrol 10 μM; Pluronic F-68 0.005% (w / v); The pH of the buffer solution is 7.4.

[0009] On the other hand, the present invention provides a method for preparing a high-efficiency electroporation buffer, which adopts the following technical solution: A method for preparing a high-efficiency electroporation buffer includes the following steps: (1) Dissolve sucrose, mannitol, trehalose, HEPES and MgCl2 in enzyme-free sterile water to prepare a basic buffer solution; (2) Prepare resveratrol stock solution and Pluronic F-68 stock solution respectively; (3) Add resveratrol stock solution and Pluronic F-68 stock solution to the basic buffer solution to achieve the above-mentioned final concentrations of resveratrol and Pluronic F-68. (4) Adjust the pH to 6.8~7.6 to obtain the electroporation buffer.

[0010] Preferably, the basic buffer solution in step (1) is formulated as follows: 220 mM sucrose, 30 mM mannitol, 20 mM trehalose, 10 mM HEPES, and 1.2 mM MgCl2.

[0011] Preferably, in step (2), the concentration of resveratrol mother liquor is 10 mM and the solvent is DMSO; the concentration of Pluronic F-68 mother liquor is 10% (w / v) and the solvent is enzyme-free sterile water.

[0012] Preferably, in step (3), the volume ratio of the basic buffer, resveratrol stock solution and Pluronic F-68 stock solution is 1000:1:0.5.

[0013] This invention also provides the application of a high-efficiency electrotransfection buffer in the electrotransfection of C2C12 mouse myoblasts.

[0014] Preferably, in electroporation, the ratio of plasmid DNA to cell number is (1~3) μg : 1×10⁻⁶. 6 Cells; the ratio of siRNA to cell number is (50~150) pmol : 1×10 6 Each cell.

[0015] Preferably, after electroporation, immediately add preheated complete culture medium at 37°C and incubate in a CO2 incubator for 5-15 minutes to allow the cells to recover.

[0016] In summary, the beneficial effects of the present invention are as follows: This invention achieves a triple balance between transfection efficiency, cell viability, and differentiation potential. By optimizing the buffer formulation (containing 220 mM sucrose, 30 mM mannitol, 20 mM trehalose, 10 mM HEPES, 1.2 mM MgCl2, and adding 10 μM resveratrol and 0.005% Pluronic F-68), the GFP-positive transfection rate of C2C12 cells reached 62.8%, an increase of 34.5 percentage points compared to the commercial control group (28.3%); while maintaining a high cell viability (OD). 450 =0.46, 24h), successfully breaking through the negative correlation bottleneck in existing technologies where "improved transfection efficiency is inevitably accompanied by cell damage"; moreover, the cells still maintain normal myogenic differentiation capacity after electroporation, and the formed myotubes are not significantly different from the control group, ensuring the reliability of downstream functional studies.

[0017] Furthermore, this invention boasts strong compatibility and significant cost advantages. This buffer is not only suitable for plasmid DNA transfection but also for transfection of small nucleic acid molecules such as siRNA, meeting various experimental needs such as gene overexpression and knockdown. In terms of cost, the total cost of preparing 100 mL of the buffer is less than 40 yuan, while the price of a commercially available product of the same volume is approximately 4000 yuan, making the cost less than one-thousandth of the latter. This significantly reduces research expenses and makes it suitable for large-scale, high-throughput experiments. Simultaneously, the buffer is easy to prepare, can be stably stored at 4℃ for one month, and can be cryopreserved long-term at -80℃, demonstrating excellent practicality and promotional value. Attached Figure Description

[0018] Figure 1 The effect of different electrotransfection buffers on the transfection efficiency of C2C12 cells; in, Figure 1 Image A shows the GFP fluorescence and bright-field diagram after electroporation of Group 1 (resveratrol added alone); Figure 1 Image B shows the GFP fluorescence and bright-field images after electroporation of Group 2 (with Pluronic F-68 added alone); Figure 1 In Figure C, GFP fluorescence and bright-field images of Group 3 (with ATP-Na2 added alone) after electroporation are shown. Figure 1 D shows the GFP fluorescence and bright-field images after electroporation of Group 4 of Example (resveratrol + Pluronic F-68 combined addition); Figure 1 E shows the GFP fluorescence and bright-field image after electroporation of the 5th group (resveratrol + ATP-Na2 combined addition); Figure 1 The image in Figure F shows the GFP fluorescence and bright-field plot after electroporation of Group 6 (Pluronic F-68 + ATP-Na2 co-addition); Figure 1 G represents the GFP fluorescence and bright-field image after electroporation of Group 7 of Example (combined addition of resveratrol, Pluronic F-68, and ATP-Na2). Figure 1 In the middle H, the GFP fluorescence and bright-field images after electroporation of the control group 1 (basal buffer) are shown. Figure 1 In Figure I, the superimposed image of GFP fluorescence and bright-field fluorescence after electroporation of the two comparative groups (formula L disclosed in the literature) is shown. Figure 1 In the middle J, the GFP fluorescence and bright-field images after electroporation of the three comparative groups (Lonza P4 commercial kit) are shown. Figure 2This is a bar chart comparing the relative cell viability of different electroporation buffers; where the horizontal axis 1-10 represent, respectively, Comparative Example 1 (basal buffer control), Comparative Example 2 (literature formulation L control), Comparative Example 3 (Lonza P4 commercial kit control), Example 1 (resveratrol alone), Example 2 (Pluronic F-68 alone), Example 3 (ATP-Na2 alone), Example 4 (resveratrol + F-68 combined), Example 5 (resveratrol + ATP-Na2 combined), Example 6 (Pluronic F-68 + ATP-Na2 combined), and Example 7 (resveratrol + Pluronic F-68 + ATP-Na2 combined). Figure 3 To compare the differentiation capacity of C2C12 cells after electroporation with different electroporation buffers; in, Figure 3 Image A is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Example 1 group (resveratrol added alone). Figure 3 Image B is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Example 2 group (with Pluronic F-68 added alone). Figure 3 Image C is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Example 3 (ATP-Na2 added alone). Figure 3 Image D is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Example 4 (resveratrol + Pluronic F-68 combined, the optimal regimen). Figure 3 Image E in the image is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Group 5 of Example (resveratrol + ATP-Na2 combined addition). Figure 3 The image in Figure F is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Group 6 of Example (Pluronic F-68 + ATP-Na2 combined addition). Figure 3 G is a bright-field micrograph of myotube formation 4 days after induction of differentiation in Example 7 (combined addition of resveratrol, Pluronic F-68, ATP, and Na2). Figure 3 In the middle, H represents a bright-field micrograph of myotube formation 4 days after induction of differentiation in control group 1 (basal buffer control). Figure 3 Figure I shows a bright-field micrograph of myotube formation 4 days after induction of differentiation in two control groups (Reference formulation L). Figure 3 J is a bright-field micrograph of myotube formation 4 days after induction of differentiation in the three control groups (Lonza P4 commercial kit control). Figure 4 Immunofluorescence staining (DAPI nuclear staining and MyHC myosin heavy chain staining) was used to compare the differentiation capacity of C2C12 cells after electroporation in Example 4 and Comparative Example 3. in, Figure 4 In the middle section, group A is the control group 3 (Lonza P4 commercial kit control) after 4 days of differentiation, MyHC myosin heavy chain staining (40×). Figure 4 In the middle B group, DAPI nuclear staining (40×) was performed on the control group 3 (Lonza P4 commercial kit control) 4 days after differentiation. Figure 4 In the middle section, group C represents the control group 3 (Lonza P4 commercial kit control) after 4 days of differentiation, with MyHC myosin heavy chain staining (40×). Figure 4 In the middle group D, the control group (Lonza P4 commercial kit control) was DAPI nuclear staining (40×) 4 days after differentiation. Figure 4 E represents MyHC myosin heavy chain staining (40×) 4 days after differentiation of Group 4 of Example (resveratrol + Pluronic F-68 combined addition). Figure 4 The middle F group (Example 4) was stained with DAPI nuclear staining (40×) 4 days after differentiation (resveratrol + Pluronic F-68). Figure 4 G represents MyHC myosin heavy chain staining (40×) 4 days after differentiation of Group 4 of Example (resveratrol + Pluronic F-68 combined addition). Figure 4 In the middle H, DAPI nuclear staining (40×) was performed 4 days after differentiation of the group of Example 4 (resveratrol + Pluronic F-68 added together). Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments.

[0020] Example Example 1

[0021] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0022] S2. Weigh 23 mg of resveratrol, dissolve it in 10 mL of DMSO, filter through 0.22 μm for sterilization, and prepare a 10 mM stock solution.

[0023] S3. Take 10 mL of the basic buffer solution prepared in step S1, add 10 μL of the resveratrol stock solution prepared in step S2, mix well, and adjust the pH to 7.4 with NaOH.

[0024] Example 2

[0025] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0026] S2. Weigh 1 g of Pluronic F-68, dissolve it in 10 mL of enzyme-free sterile water, filter it through a 0.22 μm filter to obtain a 10% (w / v) stock solution.

[0027] S3. Take 10 mL of the basic buffer solution prepared in step S1, add 5 μL of the Pluronic F-68 stock solution prepared in step S2, mix well, and adjust the pH to 7.4 with NaOH.

[0028] Example 3

[0029] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0030] S2. Weigh 0.551g of ATP-Na2, dissolve it in 10 mL of enzyme-free sterile water, and filter it through a 0.22 μm filter to obtain a 100 mM stock solution.

[0031] S3. Take 10 mL of the basic buffer solution prepared in step S1, add 100 μL of the ATP-Na2 stock solution prepared in step S2, mix well, and adjust the pH to 7.4 with NaOH.

[0032] Example 4

[0033] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0034] S2. Weigh 23 mg of resveratrol, dissolve it in 10 mL of DMSO, filter through 0.22 μm for sterilization, and prepare a 10 mM stock solution.

[0035] S3. Weigh 1 g of Pluronic F-68, dissolve it in 10 mL of enzyme-free sterile water, filter it through a 0.22 μm filter to obtain a 10% (w / v) stock solution.

[0036] S4. Take 10 mL of the basic buffer solution prepared in step S1, add 10 μL of the 10 mM resveratrol stock solution prepared in step S2 and 5 μL of the 10% Pluronic F-68 stock solution prepared in step S3, mix thoroughly, and adjust the pH to 7.4 with NaOH.

[0037] Example 5

[0038] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0039] S2. Weigh 23 mg of resveratrol, dissolve it in 10 mL of DMSO, filter through 0.22 μm for sterilization, and prepare a 10 mM stock solution.

[0040] S3. Weigh 0.551g of ATP-Na2, dissolve it in 10 mL of enzyme-free sterile water, and filter it through a 0.22 μm filter to obtain a 100 mM stock solution.

[0041] S4. Take 10 mL of the basic buffer solution prepared in step S1, add 10 μL of the 10 mM resveratrol stock solution prepared in step S2 and 5 μL of the 10% Pluronic F-68 stock solution prepared in step S3, mix thoroughly, and adjust the pH to 7.4 with NaOH.

[0042] Example 6

[0043] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0044] S2. Weigh 1 g of Pluronic F-68, dissolve it in 10 mL of enzyme-free sterile water, filter it through a 0.22 μm filter to obtain a 10% (w / v) stock solution.

[0045] S3. Weigh 0.551g of ATP-Na2, dissolve it in 10 mL of enzyme-free sterile water, and filter it through a 0.22 μm filter to obtain a 100 mM stock solution.

[0046] S4. Take 10 mL of the basic buffer solution prepared in step S1, add 5 μL of the 10% Pluronic F-68 stock solution prepared in step S2 and 100 μL of the 100 mM ATP-Na2 stock solution prepared in step S3, mix thoroughly, and adjust the pH to 7.4 with NaOH.

[0047] Example 7

[0048] The preparation steps of an electroporation buffer are as follows: S1. Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane for sterilization.

[0049] S2. Weigh 23 mg of resveratrol, dissolve it in 10 mL of DMSO, filter through 0.22 μm for sterilization, and prepare a 10 mM stock solution.

[0050] S3. Weigh 1 g of Pluronic F-68, dissolve it in 10 mL of enzyme-free sterile water, filter it through a 0.22 μm filter to obtain a 10% (w / v) stock solution.

[0051] S4. Weigh 0.551g of ATP-Na2, dissolve it in 10 mL of enzyme-free sterile water, and filter it through a 0.22 μm filter to obtain a 100 mM stock solution.

[0052] S5. Take 10 mL of the basic buffer prepared in step S1, add 10 μL of the 10 mM resveratrol stock solution prepared in step S2, 5 μL of the 10% Pluronic F-68 stock solution prepared in step S3, and 100 μL of the 100 mM ATP-Na2 stock solution prepared in step S4, mix thoroughly, and adjust the pH to 7.4 with NaOH.

[0053] Comparative Example Comparative Example 1 The preparation of a basic electroporation buffer involves the following steps: Weigh 7.53 g of sucrose, 0.55 g of mannitol, 0.68 g of trehalose, 0.24 g of HEPES, and 11 mg of MgCl2. Dissolve them in enzyme-free sterile water and bring the volume to 100 mL. After thorough shaking and dissolution, filter the solution through a 0.22 μm filter membrane to obtain the basic electroporation buffer.

[0054] Comparative Example 2 The specific steps for preparing the electroporation buffer L formulation are as follows: Preparation of S1 and Solution I: Weigh 2g of ATP-Na2 and 1.2g of MgCl2·6H2O, dissolve them in 10 mL of enzyme-free sterile water, and filter sterilize through a 0.22 μm filter; Preparation of S2 and Solution II: Weigh 6g of KH2PO4, 0.6g of NaHCO3 and 0.2g of glucose, dissolve them in 500 mL of enzyme-free sterile water, adjust the pH to 7.4 with NaOH, and sterilize by 0.22 μm filtration; S3. Mix Solution I prepared in step S1 with Solution II prepared in step S2 at a volume ratio of 1:50.

[0055] Comparative Example 3 The electroporation buffer (catalog number: PBP4-00675) provided by the Lonza P4 primary cell electroporation kit was used.

[0056] Test case Plasmid DNA was transfected into C2C12 mouse myoblasts using the electrotransfection buffers of Examples 1-7 and Comparative Examples 1-3. The specific steps are as follows: S1. Take the working solutions of the electroporation buffers of Examples 1-7 and Comparative Examples 1-3 out of 4°C and equilibrate to room temperature; preheat the complete culture medium in a 37°C water bath; check whether the Lonza 4D-Nucleofector electroporator is working properly and confirm that the program parameters are set correctly.

[0057] S2. Take C2C12 cells (ATCC) in the logarithmic growth phase, aspirate the culture supernatant, and wash 1-2 times with PBS; add an appropriate amount of trypsin (Gibco, 0.25%) for digestion, and after the cells become rounded and detach from the cell wall, add complete culture medium to stop digestion; gently pipette to prepare a single-cell suspension, transfer to a centrifuge tube, and centrifuge at 1000 g for 5 min; discard the supernatant, add 3 mL of PBS to resuspend, and gently pipette to mix; take 10 μL of the cell suspension for counting and calculate the cell density; take the cell suspension containing 2×10⁶ cells / mL.5 Centrifuge the cell suspension at 1000 g for 5 min, discard the supernatant, and retain the cell pellet for later use.

[0058] S3, S3, Take a sterile EP tube and add 17 μL of the electrotransfection buffer working solution of Examples 1-7 and Comparative Examples 1-3; Plasmid transfection: Add 0.4 μg of pmaxGFP plasmid (positive control plasmid for nuclear transfection that comes with the Lonza P4 kit, concentration 1000 ng / μL, take 0.4 μL), so that the ratio of plasmid volume (μg) to cell number (millions) is 2:1; Gently pipette and mix several times, then set aside.

[0059] S4. Add all of the above-mentioned electroporation buffer containing nucleic acid to the cell pellet prepared in step S2; gently pipette to resuspend the cell pellet to completely disperse it and form a uniform single-cell suspension; the pipetting action must be gentle to avoid generating air bubbles; air bubbles can cause electric arcs during electroporation, which seriously affects cell viability.

[0060] S5. Carefully transfer the resuspended cell suspension (approximately 20-22 μL) into the Lonza 4D electroporation strip, avoiding the introduction of air bubbles; insert the electroporation strip into the Lonza 4D-Nucleofector electroporator; set the electroporation parameters on the instrument's operating interface: Solution: Select P4 Pulse code: Select CA133 Start the electrical discharge program and wait for the instrument to indicate that the operation is complete.

[0061] S6. After electroporation, immediately remove the electroporation strip from the electroporator; quickly add 100 μL of preheated 37℃ complete culture medium (containing penicillin and streptomycin (1%)) to the electroporation strip and mix gently; place the electroporation strip in a CO2 incubator and incubate at 37℃ for 10 min for recovery; after recovery, gently aspirate all the cell suspension from the electroporation strip with a pipette and transfer it to a 12-well plate pre-filled with complete culture medium; gently shake the culture dish to distribute the cells evenly and place it in a 37℃, 5% CO2 incubator for continued culture.

[0062] S7. Twenty-four hours after electrotransfection, GFP fluorescence expression was observed under an inverted fluorescence microscope, photographs were taken, and the GFP-positive cell rate was counted. OD was measured using a CCK-8 assay kit. 450 Values ​​were used to assess cell viability; differentiation capacity was tested (if differentiation function was to be tested): when the cell confluence reached 95%~100%, the differentiation medium was replaced with one containing 2% horse serum, and the medium was changed every 2 days. After 4 days of induction of differentiation, myotube formation was observed.

[0063] Figure 1Together with Table 1, they constitute the core basis for evaluating the transfection efficiency of this invention. Figure 1 The images show superimposed bright-field micrographs of GFP fluorescence after 24 hours of electroporation in each experimental group (A-J represent Examples 1-7 and Comparative Examples 1-3, respectively), which visually reflect the differences in positive cell density among the groups. Table 1 shows the corresponding statistical results of GFP positivity rates. Both methods consistently show that Comparative Example 3 (commercial kit) had the lowest transfection efficiency at 28.3%; Comparative Example 1 (basal buffer) had 44.6%; and Comparative Example 2 (reference formulation L) had 31.0%. Based on Example 1 (basal buffer, 44.6%), the transfection efficiency was improved by adding resveratrol (Example 2, 52.3%), Pluronic F-68 (Example 3, 51.0%), and ATP-Na2 (Example 4, 50.0%) individually. When combined in pairs, Example 5 (resveratrol + F-68) had the highest efficiency of 62.8%, Example 6 (resveratrol + ATP) had 60.6%, and Example 7 (F-68 + ATP) had 57.4%. However, the efficiency of Example 8 (59.9%), which combined all three, was slightly lower than that of Example 5, indicating that there is an optimal synergistic effect between resveratrol and Pluronic F-68. Figure 1 In Example 2 (Figure D), the density of GFP-positive cells in the field of view was significantly higher than that in other groups, which was completely consistent with the highest transfection rate of 62.8% in Table 1. Figure 1 In Comparative Example 3, the fluorescent cells were the sparsest in the field of view, corresponding to the lowest transfection rate. The cross-validation of the above images and data fully demonstrates the significant advantage of the optimal formulation of the present invention (Example 5) in improving the transfection efficiency of C2C12 cells, with a 34.5 percentage point improvement in Comparative Example 3.

[0064] Table 1. Statistical analysis of GFP positivity rate 24 hours after transfection (%)

[0065] Figure 2 Together with Table 2, they constitute the core basis for evaluating cell viability in this invention. Figure 2 OD of CCK-8 method after 24 hours of electroporation in each experimental group 450 The bar chart shows the following data on the horizontal axis, from 1 to 10: Comparative Example 1 (basal buffer control), Comparative Example 2 (literature formulation L control), Comparative Example 3 (commercial kit control), Example 1 (basal buffer), Example 2 (Pluronic F-68 alone), Example 3 (ATP-Na2 alone), Example 4 (resveratrol + Pluronic F-68 combination), Example 5 (resveratrol + ATP combination), Example 6 (Pluronic F-68 + ATP combination), and Example 7 (combination of all three). Table 2 shows the corresponding OD values. 450Specific numerical values. Both showed that Comparative Example 3 had the highest survival rate (0.55), followed by Comparative Example 2 (0.50), while Comparative Example 1 had the lowest (0.38), indicating that the survival rate and transfection efficiency in the basic formulation were roughly negatively correlated. After adding each component to Example 1, except for Example 7 (OD 450 Except for OD = 0.32), the OD values ​​of the other embodiments are... 450 The values ​​were all significantly increased, indicating that resveratrol, Pluronic F-68, and ATP-Na2 not only improved transfection efficiency but also effectively protected cells from electrical damage. Most importantly, Example 4 (62.8%) showed the highest transfection efficiency. Figure 1 D) At the same time, it maintained a high cell viability rate (OD). 450 =0.46), while Example 7, despite combining all three additives, had the lowest cell survival rate (0.32), indicating that the combination of the three produced a synergistic negative effect. The above results strongly demonstrate that the optimal solution of the present invention (Example 4) has successfully broken through the long-standing bottleneck of the negative correlation between "transfection efficiency and cell survival rate" in the field of electrotransfection.

[0066] Table 2. Relative cell viability (OD) 24 h after transfection 450 )

[0067] (3) Evaluation of cell differentiation capacity Figure 3 and Figure 4 Together, they constitute the core basis for evaluating cell differentiation capacity after electroporation in this invention. Figure 3 Bright field micrographs of myotube formation 4 days after induced differentiation in each experimental group (A~J are Examples 1~7 and Comparative Examples 1~3, respectively) allow for direct observation of the differences in morphology and density of myotubes in each group. Figure 4 For comparison of immunofluorescence staining (DAPI labeling of cell nuclei, MyHC labeling of myosin heavy chains) between optimal Example 4 and Comparative Example 3, the quality of myotube formation was verified at the molecular level. Bright-field observation showed that C2C12 cells in all treatment groups were able to fuse and form typical elongated multinucleated myotube structures in differentiation medium containing 2% horse serum. Although there were slight differences in the density and thickness of myotubes among different treatment groups, Example 4 had the highest transfection efficiency. Figure 3 D) compared to the comparative example 3 with the lowest transfection efficiency ( Figure 3 Compared to J), there were no significant differences in the morphology, size, and number of myotubes. Immunofluorescence staining results further confirmed that, in comparison ratio 3 ( Figure 4 (A~D) and Example 4 ( Figure 4Both groups (E~H) showed clear MyHC positive signals uniformly distributed along the myotubes at 40× and 100× magnification. DAPI nuclear staining revealed that the cell nuclei were arranged in a chain-like pattern within the myotubes. There were no significant differences between the two groups in terms of myotube density, fusion degree, and MyHC expression intensity. These results indicate that the electrotransfection buffer of this invention achieves efficient transfection without affecting the subsequent myogenic differentiation potential of C2C12 cells, ensuring the reliability of skeletal muscle differentiation-related functional studies.

[0068] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A high-efficiency electroporation buffer, characterized in that, Components containing the following final concentrations: Sucrose 200~250 mM; Mannitol 20~40 mM; Trehalose 10~30 mM; HEPES 5~15 mM; MgCl2 0.5~2.0 mM; Resveratrol 5~20 μM; Pluronic F-68 0.001%~0.01% (w / v); The pH of the buffer solution is 6.8 to 7.

6.

2. The electroporation buffer according to claim 1, characterized in that, Components containing the following final concentrations: 220 mM sucrose; Mannitol 30 mM; Trehalose 20 mM; HEPES 10 mM; MgCl2 1.2 mM; Resveratrol 10 μM; Pluronic F-68 0.005% (w / v); The pH of the buffer solution is 7.

4.

3. A method for preparing the electroporation buffer as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Dissolve sucrose, mannitol, trehalose, HEPES and MgCl2 in enzyme-free sterile water to prepare a basic buffer solution; (2) Prepare resveratrol stock solution and Pluronic F-68 stock solution respectively; (3) Add the resveratrol stock solution and Pluronic F-68 stock solution to the basic buffer solution so that the final concentration of resveratrol and Pluronic F-68 reaches the concentration described in claim 1 or 2; (4) Adjust the pH to 6.8~7.6 to obtain the electroporation buffer.

4. The preparation method according to claim 3, characterized in that, The basic buffer solution in step (1) is formulated as follows: 220 mM sucrose, 30 mM mannitol, 20 mM trehalose, 10 mM HEPES, and 1.2 mM MgCl2.

5. The preparation method according to claim 3, characterized in that, The concentration of the resveratrol mother liquor in step (2) is 10 mM and the solvent is DMSO; the concentration of the Pluronic F-68 mother liquor is 10% (w / v) and the solvent is enzyme-free sterile water.

6. The preparation method according to claim 3, characterized in that, The volume ratio of the basic buffer, resveratrol stock solution and Pluronic F-68 stock solution in step (3) is 1000:1:0.

5.

7. The application of the electrotransfection buffer as described in claim 1 or 2 in the electrotransfection of C2C12 mouse myoblasts.

8. The application according to claim 7, characterized in that, In the electroporation process, the ratio of plasmid DNA to cell number was (1~3) μg : 1×10⁻⁶. 6 Cells; the ratio of siRNA to cell number is (50~150) pmol : 1×10 6 Each cell.

9. The application according to claim 7, characterized in that, After electroporation, immediately add preheated complete culture medium at 37°C and incubate in a CO2 incubator for 5-15 minutes to allow the cells to recover.

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