An electrotransfection method for eukaryotic cells

Abstract

The present invention relates to an electrotransfection method for eukaryotic cells. The transfection method comprises the following steps: (1) mixing a first buffer solution with a nucleic acid fragment or a plasmid containing a nucleic acid fragment to obtain a transfection gene solution; (2) mixing the cells to be transfected with a second buffer solution, standing for equilibration, centrifuging to obtain a precipitate and mixing it with the transfection gene solution, standing for incubation, and resuspending; (3) performing pulsed electric shock on the resuspended solution at 100-300 V, 80-300 μs, for 1-3 times, standing for incubation. The pH values of the first buffer solution and the second buffer solution are 7.2±0.2 respectively, the osmotic pressure of the first buffer solution is 230-280 mOsmol / kg, the osmotic pressure of the second buffer solution is 280-320 mOsmol / kg, the osmotic pressure of the first buffer solution is smaller than that of the second buffer solution, and they respectively contain glucose, threonine and / or asparagine. The present invention can better balance the cell transfection efficiency and cell survival rate.

Description

Technical Field

[0001] The present invention relates to the field of eukaryotic cell gene transfection, and particularly to an electrotransfection method for eukaryotic cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are stem cells derived from the mesoderm with multi-directional differentiation ability. They mainly exist in the connective tissues and organ interstitium throughout the body, and are most abundant in bone marrow tissue. They are adult stem cells with self-renewal and multi-directional differentiation potential. Bone marrow MSCs are easily obtained by in vitro isolation and culture, and have low cell immunogenicity, and have become ideal seed cells in tissue engineering, gene therapy and regenerative medicine research.

[0003] When mesenchymal stem cells are used in biological research such as gene editing, gene function research, gene expression regulation, mutation analysis and protein production, foreign molecules such as DNA and RNA need to be introduced into mesenchymal stem cells by transfection. Common cell transfection methods mainly include: virus infection method, liposome transfection method, electroporation transfection method, microinjection method, calcium phosphate co-precipitation method, cationic substance-mediated transfection and other methods. Different transfection methods have their own advantages and disadvantages:

[0004] Liposome transfection method: Cationic liposomes with a positive charge on the surface act on the phosphate group of nucleic acid through electrostatic interaction, and then encapsulate the DNA molecule to form a DNA-liposome complex. The cell membrane surface is negatively charged and can adsorb the liposome complex, and then enter the cell through membrane fusion or endocytosis. It is suitable for gene transfection of suspended or adherent cultured cells, with convenient operation and no need for special equipment, etc., and its transfection rate is relatively high. Its disadvantage is that liposomes have certain toxicity to cells, and the transfection time generally does not exceed 60 hours, which has potential safety hazards for gene introduction of clinical application cells.

[0005] Virus infection: It can quickly and efficiently infect cells, and the infection efficiency can be as high as 100%, and the transfection success rate is high. Its disadvantages are that virus packaging is required to obtain highly titrated infectious viruses; virus infection has a certain impact on cell viability, and at the same time, the virus vector genome may integrate into the genome. The virus transfection method cannot guarantee its safety and cannot be used clinically, which has limitations.

[0006] Electroporation transfection method: Through physical methods, the permeability of the cell membrane is changed. By applying an electric shock, the cell membrane can be reversibly perforated to form transient water channels, or the pores on the membrane can be opened, promoting the diffusion of DNA molecules into the cell. Its advantage is that the risk for clinical cell applications is relatively low, there is no risk of exogenous gene introduction other than the introduced fragment, and there is no toxicity risk of compounds such as liposomes. It can achieve the introduction of relatively large fragment genes. However, its disadvantage is that a high electric field strength will kill 50%-70% of the cells, and the transfection efficiency is relatively low. Therefore, professional transfection reagents are needed to protect the cells, reduce the cell death rate, and at the same time improve the transfection efficiency.

[0007] For the transfection of eukaryotic cells, such as human mesenchymal stem cells, the most ideal method should be one that can simultaneously achieve high transfection efficiency and low cell death rate. Summary of the Invention

[0008] The purpose of the present invention is to provide an electroporation method for eukaryotic cells that can improve the cell transfection efficiency and at the same time reduce the cell death rate.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is:

[0010] An electroporation method for eukaryotic cells, the transfection method comprising the following steps:

[0011] (1) Mix a first buffer solution with a nucleic acid fragment or a plasmid containing a nucleic acid fragment to obtain a transfection gene solution;

[0012] (2) Mix the cells to be transfected with a second buffer solution, let it stand for equilibration, centrifuge to obtain a precipitate, mix the precipitate and the transfection gene solution, let it stand for incubation, and resuspend;

[0013] (3) Electroporate the resuspended solution, and the electroporation parameters are: 100-300 v, 100-300 μs, 1-3 pulses. After electroporation, let it stand for incubation,

[0014] Wherein, the pH values of the first buffer solution and the second buffer solution are independently 7.2±0.2,

[0015] The osmotic pressure of the first buffer solution is 230-280 mOsmol / kg, the osmotic pressure of the second buffer solution is 280-320 mOsmol / kg, and the osmotic pressure of the first buffer solution is smaller than that of the second buffer solution,

[0016] The first buffer solution and the second buffer solution independently contain 0.8-1.2 g / L glucose and 0.3-0.5 g / L amino acid, and the amino acids in the first buffer solution and the second buffer solution are independently threonine and / or asparagine.

[0017] Preferably, the osmotic pressure of the first buffer is 250-270 mOsmol / kg, and the osmotic pressure of the second buffer is 285-300 mOsmol / kg.

[0018] Preferably, the components of the first buffer include: 2-2.5 g / L potassium chloride, 0.05-0.1 g / L potassium dihydrogen phosphate, 0.1-0.15 g / L dipotassium hydrogen phosphate, 28-32 g / L inositol, 0.8-1.2 g / L glucose, 0.3-0.4 g / L threonine, 0.08-0.12 g / L asparagine, and the balance is injection water.

[0019] Preferably, the components of the second buffer include: 2-2.5 g / L potassium chloride, 0.05-0.1 g / L potassium dihydrogen phosphate, 0.1-0.15 g / L dipotassium hydrogen phosphate, 37-40 g / L inositol, 0.8-1.2 g / L glucose, 0.3-0.4 g / L threonine, 0.08-0.12 g / L asparagine, and the balance is injection water.

[0020] Preferably, the electroshock parameters are: 150-200 v, 150-250 μs, and 1-2 pulses.

[0021] Preferably, in step (1), the nucleic acid fragment or the plasmid containing the nucleic acid fragment is diluted to 0.2-10 μg / mL with the first buffer dye solution.

[0022] Preferably, in step (2), the cells to be transfected are mixed with the second buffer at a concentration of 1×10 6～ 5×10 6 cells / mL, allowed to stand and equilibrate for 1-3 min, centrifuged at a rotation speed of 800-1200 r / min, the precipitate is collected and mixed with the above transfection gene solution, incubated for 1-3 min, resuspended and transferred to a transfection cup.

[0023] Preferably, before transfection, the cells to be transfected are cultured: before transfection, the cells to be transfected are passaged or resuscitated and inoculated into a serum-free medium, and the inoculation density is 1×10 4 ~5×10 4 / cm 2 , cultured at 37°C and 5% CO2 for 48-72 h, the cells grow to 70%-85% confluence, and the cells in the logarithmic growth phase of proliferation are collected for the above electrotransfection.

[0024] In the present invention, the nucleic acid is a DNA fragment and / or an RNA fragment.

[0025] According to some specific embodiments, the cells to be transfected are human mesenchymal stem cells.

[0026] More specifically, the components of the first buffer solution include: 2.2365 g / L potassium chloride, 0.0871 g / L potassium dihydrogen phosphate, 0.1361 g / L dipotassium hydrogen phosphate, 29 g / L inositol, 1 g / L glucose, 0.35 g / L threonine, 0.1 g / L asparagine, and the balance is injection water. The osmotic pressure of the first buffer solution is 261 mOsmol / kg.

[0027] More specifically, the components of the second buffer solution include: 2.2365 g / L potassium chloride, 0.0871 g / L potassium dihydrogen phosphate, 0.1361 g / L dipotassium hydrogen phosphate, 38.2 g / L inositol, 1 g / L glucose, 0.35 g / L threonine, 0.1 g / L asparagine, and the balance is injection water. The osmotic pressure of the second buffer solution is approximately 289 mOsmol / kg.

[0028] More specifically, the electroshock parameters are: 180 v, 200 μs, and 2 pulses.

[0029] The present invention also provides a eukaryotic cell electroporation reagent, which includes the first buffer solution and the second buffer solution used in the above-mentioned eukaryotic cell electroporation method.

[0030] In the present invention, the cells to be transfected are equilibrated in the second buffer solution and then enter the first buffer solution. The change in the cell surrounding environment is gentle, effectively reducing the cell death rate. Moreover, by optimizing the formulations of the first buffer solution and the second buffer solution, it is beneficial to protect the cells from adapting to the change in membrane osmotic pressure while improving the transfection efficiency and reducing the cell death rate.

[0031] Due to the implementation of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0032] The method of the present invention can reduce the cell death rate while improving the transfection effect of eukaryotic cells, and the positive rate of the transfected eukaryotic cells is higher. Description of the Drawings

[0033] Figure 1 It is a schematic diagram of the cell fluorescence expression after transfection of human bone marrow mesenchymal stem cells in the embodiment, where the left figure is the fluorescence photography mode and the right figure is the white light photography mode. Detailed Embodiments

[0034] The present invention will be further described below through specific embodiments, but it is not a limitation to the present invention, only for illustrative purposes. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific uses, and the implementation conditions not specified are the conventional conditions in the industry.

[0035] In order to solve the problem that the existing cell electroporation methods cannot take into account both transfection efficiency and cell survival rate, resulting in poor electroporation effect of human mesenchymal stem cells. The present invention discloses an improved electroporation method for human mesenchymal stem cells, a transfection protectant and a second buffer solution for use in conjunction with this method.

[0036] According to the electroporation method for human mesenchymal stem cells of the present invention, a transfection protectant for use in conjunction is designed. The transfection protectant includes a first buffer solution and a second buffer solution. Under the electric shock conditions: 100 - 300v, 80 - 300μs, 1 - 2 pulses, better electroporation efficiency and lower cell death rate can be obtained.

[0037] According to the embodiments of the present invention, the preferred formulation of the first buffer solution is: 2 - 2.5 g / L potassium chloride, 0.05 - 0.1 g / L potassium dihydrogen phosphate, 0.1 - 0.15 g / L dipotassium hydrogen phosphate, 28 - 32 g / L inositol, 0.8 - 1.2 g / L glucose, 0.3 - 0.4 g / L threonine, 0.08 - 0.12 g / L asparagine, with the balance being injection water.

[0038] The preferred formulation of the second buffer solution is: 2 - 2.5 g / L potassium chloride, 0.05 - 0.1 g / L potassium dihydrogen phosphate, 0.1 - 0.15 g / L dipotassium hydrogen phosphate, 37 - 40 g / L inositol, 0.8 - 1.2 g / L glucose, 0.3 - 0.4 g / L threonine, 0.08 - 0.12 g / L asparagine, with the balance being injection water.

[0039] The first buffer solution and the second buffer solution are respectively filtered and sterilized through a 0.22μm sterile filter and stored for standby at 2 - 8°C.

[0040] The method for highly efficient electroporation of human mesenchymal stem cells provided by the present invention includes the following steps:

[0041] (1) Dilute nucleic acid fragments or plasmids containing nucleic acid fragments to 0.2 - 10 μg / mL with the first buffer dye solution to obtain a transfected gene solution;

[0042] (2) Mix human mesenchymal stem cells and the second buffer solution at a concentration of 1×10 6～ 5×10 6 cells / mL, let stand for equilibration for 1 - 3 min, centrifuge at a rotation speed of 800 - 1200 r / min, collect the precipitate and mix it with the above-mentioned transfected gene solution, incubate for 1 - 3 min, resuspend and transfer to a transfection cup;

[0043] (3) Perform electrotransfection, let stand and incubate for 1 - 3 min, and the electroshock parameters are: 100 - 300v, 80 - 300μs, 1 - 3 pulses.

[0044] Preferably, before transfection, the cells to be transfected are cultured: before transfection, passage or resuscitate and inoculate human mesenchymal stem cells, and the inoculation density is 1×10 4 ~5×10 4 / cm 2 , use the MSC serum-free amplification kit C011 (Excell Bio, ME000-C011), culture at 37°C and 5% CO2 for 48-72 h, when the cells grow to 70-85% confluence, collect the cells in the logarithmic growth phase for transfection.

[0045] Preferably, after transfection, the transfected human mesenchymal stem cells are aspirated out, slowly added to the pre-equilibrated complete medium, shaken well, and placed in an incubator at 37°C for 6-48 hours to observe or test the transfection effect.

[0046] The present invention can rapidly introduce gene sequences into human mesenchymal stem cells, avoid the cytotoxicity caused by liposome transfection, and avoid the potential risks of virus transfection. It can efficiently introduce the target gene into the genome of human mesenchymal stem cells in a physical manner, realizing the introduction of foreign genes or the integration of gene fragments into the genome.

[0047] The present invention has the following advantages:

[0048] (1) The method of the present invention realizes the rapid introduction of plasmids or gene fragments through electroporation, with a short operation time, reducing the cytotoxic action time of liposomes, viruses, etc. It can more efficiently integrate the target gene into the eukaryotic cell genome, and can reduce the later drug screening time;

[0049] (2) For the size of gene fragments transfected by liposome transfection and virus transfection methods, it is affected by the carrying capacity of liposomes and virus vectors. Liposome transfection is suitable for the transfection of gene fragments below 10 Kb, and virus transfection is suitable for gene fragments below 8 Kb. The electroporation method can achieve the transfection of gene fragments of 1 Kb-15 Kb. Existing technologies usually require a voltage of more than 500 V to achieve a higher transfection effect, but the cell mortality rate is also higher. The electroporation method of the present invention uses the first buffer solution and the first buffer solution with different osmotic pressures designed with different materials, and the change of the cell surrounding environment is gentle, which is beneficial to protecting the cells to adapt to the change of membrane osmotic pressure, and can better tolerate the electric shock conditions. When the transfection voltage is 180 V, it can tolerate 100-300 μs and can tolerate 2 pulse electric shocks. Taking human mesenchymal stem cells as an example, the transfection efficiency can reach 67%, and at the same time the cell mortality rate is reduced to 33%. It can achieve the efficient introduction of foreign genes at a lower voltage. The transfection method of the present invention is more suitable for gene modification or research of cells related to clinical research. Compared with virus transfection, it does not introduce foreign sequences other than the target gene, and compared with liposome transfection, it does not contain components that change the membrane structure or are toxic to cells, and is safer and has lower risks.

[0050] In the following examples, unless otherwise specified, the experimental methods used are conventional methods in the art, and the reagents used are commercially available products.

[0051] The human mesenchymal stem cells used in the following examples were purchased from the United States (ATCC, PCS-500-012, batch number: 70011720); the exogenous gene was the linearized plasmid vector PLMN containing EGFP (constructed by the Embryo Engineering Laboratory of Yangzhou University, Jiangsu Province, containing the green fluorescent protein reporter gene, and the vector size was 15 KB), and the purified quantitative concentration was 498 ng / μL; the transfection equipment was electroporation cuvettes (purchased from Eppendorf). In other examples, other commercially available plasmids can also be used.

[0052] Culture of human bone marrow mesenchymal stem cells to be transfected:

[0053] According to the conventional cell culture operation, resuscitate the P4 generation of human bone marrow mesenchymal stem cells, and inoculate them into a T25 culture flask at a density of 3×10 4 / cm 2 , and culture them with DMEM medium containing 10% fetal bovine serum. Culture them under the conditions of 37°C, 5% CO2, and saturated humidity for about 72 h, and passage culture when the confluence reaches 80%; passage inoculate according to a ratio of 1:3, change the medium after 24 h, observe after 48 h, harvest the cells when the cells grow to about 75% confluence, and prepare for transfection experiments.

[0054] Prepare transfection protectant:

[0055] Prepare the first buffer and the second buffer for Examples 1 to 6, Comparative Example 1 and Comparative Example 2 according to the formula in Table 1.

[0056] Table 1

[0057]

[0058] First, weigh potassium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, dissolve them with injection water, then divide them into multiple portions, and add inositol, glucose, threonine, and / or asparagine respectively. Adjust the pH to 7.0 - 7.4, filter and sterilize with a 0.22 μm sterile filter, and store at 2 - 8°C for later use. The weighing unit of each component is g / L.

[0059] The osmotic pressure of the first buffer solution in Example 1 was 261 mOsmol / kg, and the osmotic pressure of the second buffer solution was approximately 289 mOsmol / kg; the osmotic pressure of the first buffer solution in Comparative Example 1 was 217 mOsmol / kg, and the osmotic pressure of the second buffer solution was 261 mOsmol / kg; the osmotic pressure of the first buffer solution in Comparative Example 2 was 252 mOsmol / kg, and the osmotic pressure of the second buffer solution was 280 mOsmol / kg.

[0060] Transfection:

[0061] (1) Preparation of transfection solution:

[0062] Take out the pre-purified and quantified transfection gene (plasmid vector PLMN, quantified concentration of 498 ng / μL);

[0063] Take 24 μL of the transfection gene lysate and add it to 1176 μL of the first buffer solution of each of the above examples and comparative examples, mix well and set aside.

[0064] (2) Cell transfection:

[0065] Collect human bone marrow mesenchymal stem cells in the logarithmic growth phase of proliferation. After resuspending and washing with PBS, perform cell counting. Take 1×10 6 cells into a sample tube, centrifuge to collect the cells, then add 1 mL of the second buffer solution of the example or comparative example to resuspend, mix well and let stand for 1 min for equilibration, then centrifuge at 1000 rp / min for 5 min, collect the cells, discard the supernatant, add 400 μL of the transfection solution corresponding to the transfection equilibration solution prepared in step (1). Three replicates are set for each of the examples and comparative examples. Incubate at room temperature for 1 min. After the incubation, gently pipette to resuspend the cells and transfer them into a transfection cup. Set the transfection instrument, and the transfection electric shock conditions are: 180 v, 200 μs, 2 pulses; perform electric shock, and take out and let stand for 3 min after the electric shock.

[0066] Culture and identification of human bone marrow mesenchymal stem cells after electroporation:

[0067] Aspirate the transfected cells, slowly add them to the pre-equilibrated complete medium, shake well, take samples for analysis, inoculate the remaining cells into a cell culture plate, culture at 37 °C, 5% CO2, and saturated humidity for 24 h, collect the supernatant, perform cell counting, and calculate the number of dead cells transfected by the cells; culture at 37 °C, 5% CO2, and saturated humidity for 48 hours, digest the cells for flow cytometry detection, record the number of positive cells, and the detection results are shown in Table 2.

[0068] Table 2

[0069]

[0070] Table 2 results show that under fixed transfection conditions (180v, 200μs, 2 pulses), the use of the first buffer solution and the second buffer solution of Comparative Example 1 can achieve the introduction of foreign genes (see the electron microscopy results in Figure 1 ). After transfection, the highest cell viability can reach 67%, the mortality rate can be maintained at 33%, and the positive rate can reach 67% 48 hours after transfection, which is significantly higher than that of other examples. Compared with Example 1, Comparative Example 1 and Comparative Example 2 did not use glucose, threonine, and asparagine. The osmotic pressure of Comparative Example 1 was too low to obtain good transfection efficiency and the cell mortality rate was very high. In Comparative Example 2, inositol was added to adjust the osmotic pressure to 280 mOsmol / kg. Compared with Comparative Example 1, although the cell viability could be increased to 41%, the transfection efficiency was only 15% under this electroshock condition. It can be seen that the osmotic pressure and raw material compounding of the first buffer solution and the second buffer solution jointly affect the transfection effect of human bone marrow mesenchymal stem cells.

[0071] Effect of Different Electroporation Conditions on Transfection Efficiency of Human Bone Marrow Mesenchymal Stem Cells

[0072] The first buffer solution and the second buffer solution used for testing and the electroshock conditions used are shown in Table 3. Among them, the commercially available electroporation solution is Gibco, A4997901. The transfection method and the testing method are the same as above and will not be elaborated here. The test results are shown in Table 4.

[0073] Table 3

[0074]

[0075] Table 4

[0076]

[0077]

[0078] As shown in Table 4, increasing the voltage, prolonging the pulse time, and increasing the number of pulses can all increase the transfection positive rate to varying degrees. However, the increase of these conditions also increases the damage to human bone marrow mesenchymal stem cells and reduces the cell viability. In order to achieve the highest transfection positive rate under a suitable cell viability, after various screenings, the electroshock conditions of Test Example 1 were preferably selected as the transfection conditions under the protection solution conditions of this case. Transfection voltages above 390V increase the damage to cells and reduce the cell viability after transfection. The transfection effect of using the first buffer solution and the second buffer solution of Example 1 in combination with the electroshock conditions of Test 1 is the best. Better transfection effects can also be obtained at lower voltages and the number of electroshocks. The cell activity after transfection is significantly higher than that of the Gibco electroporation solution, the cell transfection mortality rate is significantly lower than that of the Gibco electroporation solution, and the positive rate is significantly higher than that of the Gibco electroporation solution.

[0079] The above examples are only exemplary embodiments of the present invention and are not used to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions within the essence and protection scope of the present invention, and such modifications or equivalent substitutions should also be regarded as falling within the protection scope of the present invention.

Claims

1. An electrotransfection method for eukaryotic cells, characterized in that, The transfection method described above includes the following steps: (1) Mix a first buffer with a nucleic acid fragment or a plasmid containing a nucleic acid fragment to obtain a transfection gene solution; (2) Mix the cells to be transfected with a second buffer, let it stand for equilibration, centrifuge to obtain a precipitate, mix the precipitate and the transfection gene solution, let it stand for incubation, and gently resuspend the cells with a pipette; (3) Perform electroshock on the resuspended solution. The electroshock parameters are: 100 - 300 v, 80 - 300 μs, 1 - 3 pulses. After electroshock, let it stand for incubation. Among them, the pH values of the first buffer and the second buffer are independently 7.2 ± 0.

2. The osmotic pressure of the first buffer is 250 - 270 mOsmol / kg, and the osmotic pressure of the second buffer is 285 - 300 mOsmol / kg. The components of the first buffer include: 2 - 2.5 g / L potassium chloride, 0.05 - 0.1 g / L potassium dihydrogen phosphate, 0.1 - 0.15 g / L dipotassium hydrogen phosphate, 28 - 32 g / L inositol, 0.8 - 1.2 g / L glucose, 0.3 - 0.4 g / L threonine, 0.08 - 0.12 g / L asparagine, and the balance is injection water. The components of the second buffer include: 2 - 2.5 g / L potassium chloride, 0.05 - 0.1 g / L potassium dihydrogen phosphate, 0.1 - 0.15 g / L dipotassium hydrogen phosphate, 37 - 40 g / L inositol, 0.8 - 1.2 g / L glucose, 0.3 - 0.4 g / L threonine, 0.08 - 0.12 g / L asparagine, and the balance is injection water.

2. The electrotransfection method for eukaryotic cells according to claim 1, characterized in that, The electroshock parameters are: 150 - 200 v, 150 - 250 μs, 2 pulses.

3. The electrotransfection method for eukaryotic cells according to claim 1, characterized in that, In step (1), dilute the nucleic acid fragment or the plasmid containing the nucleic acid fragment to 0.2 - 10 μg / mL with the first buffer solution.

4. The electrotransfection method for eukaryotic cells according to claim 1, characterized in that, In step (2), mix the cells to be transfected with the second buffer at a concentration of 1×10 6～ 5×10 6 cells / mL, let stand for equilibration for 1 - 3 min, centrifuge at a rotation speed of 800 - 1200 r / min, collect the precipitate and mix it with the above transfection gene solution, incubate for 1 - 3 min, resuspend and transfer to a transfection cup.

5. The electrotransfection method for eukaryotic cells according to claim 1, characterized in that, Before transfection, culture the cells to be transfected: Before transfection, passage or resuscitate and inoculate the cells to be transfected into a serum-free medium at an inoculation density of 1×10 4 ~5×10 4 / cm 2 , culture at 37°C and 5% CO2 for 48 - 72 h until the cells grow to 70% - 85% confluence, and collect the cells in the logarithmic growth phase of proliferation for the electroporation transfection.

6. The electrotransfection method for eukaryotic cells according to claim 1, characterized in that, The cells to be transfected are human mesenchymal stem cells; the nucleic acid is a DNA fragment and / or an RNA fragment.

7. The electrotransfection method for eukaryotic cells according to claim 1 or 6, characterized in that, The components of the first buffer include: 2.2365 g / L potassium chloride, 0.0871 g / L potassium dihydrogen phosphate, 0.1361 g / L dipotassium hydrogen phosphate, 29 g / L inositol, 1 g / L glucose, 0.35 g / L threonine, 0.1 g / L asparagine, and the balance is injection water. The osmotic pressure of the first buffer is 261 mOsmol / kg. The components of the second buffer include: 2.2365 g / L potassium chloride, 0.0871 g / L potassium dihydrogen phosphate, 0.1361 g / L dipotassium hydrogen phosphate, 38.2 g / L inositol, 1 g / L glucose, 0.35 g / L threonine, 0.1 g / L asparagine, and the balance is injection water. The osmotic pressure of the second buffer is approximately 289 mOsmol / kg. The electroshock parameters are: 180 v, 200 μs, 2 pulses.

8. An electrotransfection reagent for eukaryotic cells, characterized in that, The eukaryotic cell electroporation reagent includes the first buffer and the second buffer used in the eukaryotic cell electroporation method described in any one of claims 1 - 7.

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