Electro-fusion buffer solution for preparing hybridoma cells and application of electro-fusion buffer solution

Abstract

The invention provides an electrofusion buffer solution for preparing hybridoma cells and application of the electrofusion buffer solution. The electrofusion buffer solution disclosed by the invention is prepared from the following components: 100 to 300 mM of saccharides, 0.1 to 0.2 mM of (CH3COO) 2Ca. H2O, 0.1 to 0.2 mM of (CH3COO) 2Mg. 4H2O and 0.05 to 0.2 percent of BSA (Bovine Serum Albumin). The components and proportion of the electrofusion buffer solution are optimized, the stability and compatibility among the components are ensured, the electric conductivity of cells in the electrofusion process is reduced, the stability of the cells in an electric field environment is improved, the cells can be stably arranged and fused, the fusion efficiency of the cells is improved, and the cell activity is kept; meanwhile, the cost required by the experiment process is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of hybridoma cell production and application, and in particular relates to an electrofusion buffer for hybridoma cell preparation and application thereof. Background Art

[0002] Hybridoma technology, also known as monoclonal antibody technology, refers to the technology of fusing immune B lymphocytes and mouse myeloma cells to obtain single clone cells, thereby forming hybridoma cells that can secrete a highly pure monoclonal antibody. The basis of this technology is cell fusion technology. The common cell fusion method is electrofusion. Cell electrofusion technology is a new type of fusion-promoting technology established in the 1980s. Its principle is to place the mixture of two cells in a low-voltage AC electric field to aggregate the cells into beads, and then apply high-voltage electric pulses to promote cell fusion. This technology is simple to operate, has relatively low toxicity to cells, can induce cell fusion and directly select hybrid cells under direct microscope, and has a high cell fusion rate. However, the direct current shock during the application of voltage can easily lead to cell death, or lead to a small number of surviving cells and poor generation efficiency, resulting in failure of electrofusion.

[0003] In order to improve the cloning efficiency, increase the success rate of obtaining specific antibodies, and obtain high yields of antibodies, the existing technology adjusts the culture medium of hybridoma cells. CN 103865880A discloses a culture medium and a preparation method thereof, including 1640 complete culture medium and adding recombinant human IL-21, which can promote the growth of newly generated hybridoma cells and enhance the ability of hybridoma cells to secrete specific antibodies. However, this method requires improving the culture environment of hybridoma cells after they are generated, and is essentially still limited by cell fusion efficiency and cell survival rate.

[0004] In order to improve the efficiency of cell fusion and cell survival rate, the cells can be properly treated with hypotonicity and certain protective agents can be added before applying electric pulses. This can not only reduce the conductivity of the cells and provide a stable fusion environment for cell electrofusion, but also significantly improve the efficiency of electrofusion while maintaining cell activity, laying the foundation for subsequent cell secretion of highly specific antibodies and expanded culture. Therefore, finding an electrofusion buffer for hybridoma cell preparation is a key to improving cell fusion efficiency and cell survival rate. Summary of the invention

[0005] The present application provides an electrofusion buffer for hybridoma cell preparation and its application, so as to improve the fusion efficiency and cell viability of hybridoma cells prepared by electrofusion method and maximize the positive rate of screening items.

[0006] In order to solve the above technical problems, one of the purposes of the present invention is to provide an electrofusion buffer for hybridoma cell preparation, comprising the following components: 100-300 mM carbohydrates, 0.1-0.2 mM (CH3COO)2Ca·H2O, 0.1-0.2 mM (CH3COO)2Mg·4H2O, and 0.05-0.2% BSA.

[0007] Furthermore, the electrofusion buffer is prepared by the following steps: According to the electrofusion buffer formula, each component was weighed according to the ratio, and then the weighed sugars, (CH3COO)2Ca·H2O, (CH3COO)2Mg·4H2O and BSA were fully dissolved in ultrapure water in turn. After measuring the pH value, the volume was adjusted to 1000 mL, filtered and sterilized with a 0.22 μm filter membrane, and stored at 4°C for later use.

[0008] Furthermore, the sugar is any one of glucose, D-trehalose or sucrose.

[0009] Furthermore, the pH of the electrofusion buffer is 7.0-7.2.

[0010] In the electrofusion buffer described in the present invention, sugars are used as protective agents to maintain the osmotic pressure of cells and form a unique protective film, reduce the damage to cells in the electrolyte solution and during the electrofusion process, and improve the survival rate of cells; (CH3COO)2Ca·H2O and (CH3COO)2Mg·4H2O have relatively weak conductivity, which can reduce damage to cells; and bovine serum albumin (BSA) can provide nutrients for cells, has the function of protecting the cytoplasm, and improves the efficiency and survival rate of cells during cell fusion. Therefore, the synergistic effect between the components in the electrofusion buffer described in the present invention can effectively maintain the osmotic pressure of fused cells, form a unique protective film on the cell surface, so that the fused cells are protected and quickly repaired in the electrolyte matrix and during the direct current voltage electroporation, which is beneficial to the survival and subsequent proliferation of hybridoma cells.

[0011] In order to solve the above technical problems, the second object of the present invention is to apply the electrofusion buffer to the hybridoma technology. Specifically, it is a method for preparing hybridoma cells by applying the electrofusion buffer, characterized in that the method comprises the following steps: (1) Prepare mouse spleen cell suspension, wash and count, and mix mouse spleen cells with pre-cultured SP2 / 0 myeloma cells in a ratio of 2:1–5:1; (2) resuspending the cells in step (1) in electrofusion buffer and washing them twice by centrifugation using the buffer; (3) adding the cell suspension from step (2) into the fusion chamber of the electrofusion instrument which has been pre-rinsed with buffer; (4) fusing spleen cells with SP2 / 0 myeloma cells according to an electrofusion procedure to form a fusion product containing hybridoma cells; (5) Add the cell suspension after electrofusion to HAT medium and incubate in a 37°C incubator for 60 min to allow the cells to adapt and recover; (6) Add the fused cell suspension to a 96-well plate containing feeder cells for culture and subsequent screening.

[0012] The present invention applies the electrofusion buffer to the preparation process of hybridoma cells, and uses the electrofusion buffer to centrifuge and wash the mixed cells of mouse spleen cells and SP2 / 0 myeloma cells twice, which can more effectively wash away impurities in the mixed cells and relieve their contact inhibition. During the electrofusion process, the electrofusion buffer can fully disperse the cells, while ensuring the rapid transport of cell nutrients and reducing cell electric shock damage. After the cell suspension after electrofusion is transferred to the HAT culture medium and incubated in a 37°C incubator for 60 min, the cells can adapt and recover, and promote the growth of newly generated hybridoma cells in the later stage. The method effectively improves the cloning efficiency and the success rate of obtaining specific antibodies.

[0013] Furthermore, the cell fusion density was controlled at 0.5-1×10 7 Pieces / mL.

[0014] Furthermore, the electric fusion procedure is completed by operating the BTX ECM2001+ fusion instrument. Specifically, its parameters are set as follows: the AC voltage parameters are: the first AC electric field is: initial voltage VO1: 30 V, peak voltage VF1: 50 V, AC electric field frequency 2 MHz, and continuous action time is 20 seconds; the second AC electric field is: initial voltage VO2: 60V, peak voltage VF2: 60 V, AC electric field frequency 2 MHz, and continuous action time is 20 seconds.

[0015] The parameters of the subsequent DC pulses were: DC pulse voltage 600 V, action time 50 microseconds, and pulse number 1; the initial voltage VO3 of the last AC electric field: 50 V, peak voltage VF3: 5 V, AC electric field frequency 0.8 MHz, and continuous action time of 7 seconds.

[0016] Beneficial effects of the present invention: The present invention forms cells into a fully arranged pearl chain through the synergistic effect between the components and two alternating current pre-arrangement procedures, thereby enhancing the contact area between cells, effectively improving the activity, fusion efficiency and cell fusion rate of cells during the electrofusion process, and generally improving the hybridoma clone formation rate and viability, which is beneficial to the screening of positive hybridomas. The novel electrofusion buffer can effectively maintain the osmotic pressure of fused cells, form a unique protective film on the cell surface, and protect and quickly repair the fused cells in the electrolyte matrix and during the direct current voltage electroporation, which is beneficial to the survival and subsequent proliferation of hybridoma cells. At the same time, it can reduce costs. The cost required by the present invention is much lower than that of commercially available electrofusion buffer products, achieving the effect of reducing costs and increasing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0018] Figure 1 This is a bar graph comparing cell fusion efficiency in Example 4 of the present application; Figure 2 A line-bar graph comparing cell survival rate and cell number in Example 4 of the present application; Figure 3 This is a bar graph comparing the number of positive wells in Example 4 of the present application; DETAILED DESCRIPTION The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The instruments, reagents and methods used in the following examples are commonly used in the art unless otherwise specified.

[0020] Example 1 The invention provides an electrofusion buffer for preparing hybridoma cells, comprising the following components: 100-300 mM sugar, 0.1-0.2 mM (CH3COO)2Ca·H2O, 0.1-0.2 mM (CH3COO)2Mg·4H2O, and 0.05-0.2% BSA.

[0021] The following will study the preparation of electrofusion buffer with different sugar components and its cell fusion efficiency, viability and screening of positive wells The electrofusion buffers of different sugar components of the present invention are prepared as follows: Solution A. Glucose composition solution: 300 mM glucose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, 0.1% BSA Step 1: Weigh 300 mM glucose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0022] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0023] Step 3: Sterilize the glucose electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0024] Scheme B. D-trehalose Composition: 300 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, 0.1% BSA Step 1: Weigh 300 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O and 0.1% BSA.

[0025] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0026] Step 3: Sterilize the D-trehalose electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0027] Scheme C. Sucrose composition: 300 mM sucrose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O and 0.1% BSA Step 1: Weigh 300 mM sucrose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0028] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0029] Step 3: Filter the sucrose electrofusion buffer through a 0.22 μm filter membrane to sterilize it and store it at 4°C until use.

[0030] The experimental results and data are shown in Table 1: Table 1 From the above experimental results and data, it can be seen that by comparing the cell fusion rate, number of positive holes, number of cells and viability after fusion of electrofusion buffers prepared with different sugars, it is found that the electrofusion buffer prepared with D-trehalose is superior. This is because trehalose can better maintain the osmotic pressure of cells and form a unique protective membrane, reducing the damage to cells during electrofusion, thereby improving the cell fusion efficiency and the number of positive holes.

[0031] Example 2 The invention provides an electrofusion buffer for preparing hybridoma cells, comprising the following components: 100-300 mM sugar, 0.1-0.2 mM (CH3COO)2Ca·H2O, 0.1-0.2 mM (CH3COO)2Mg·4H2O, and 0.05-0.2% BSA.

[0032] The following will study the preparation of high, medium and low sugar concentration electrofusion buffer and their cell fusion efficiency, viability and screening of positive wells The high and low concentration electrofusion buffers of the present invention are configured as follows: Protocol A. High-sugar protocol: 300 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA Step 1: Weigh 300 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0033] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0034] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0035] Solution B. Medium sugar solution: 200 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA Step 1: Weigh 200 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0036] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0037] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0038] Scheme C. Low-sugar solution: 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA Step 1: Weigh 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0039] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0040] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0041] The experimental results and data are shown in Table 2: Table 2 Based on the above experimental results and data, by comparing the cell fusion rate, number of positive wells, number of cells and viability after fusion in electrofusion buffers prepared with different concentrations of trehalose, it was found that the electrofusion buffer prepared with low concentrations of trehalose has better effect. This is because cells can quickly restore spherical shape in hypotonic solution, which is convenient for observing the fusion process, and can also reduce cell conductivity.

[0042] Example 3 The invention provides an electrofusion buffer for preparing hybridoma cells, comprising the following components: 100-300 mM sugar, 0.1-0.2 mM (CH3COO)2Ca·H2O, 0.1-0.2 mM (CH3COO)2Mg·4H2O, and 0.05-0.2% BSA.

[0043] The following will study the preparation of different concentrations of BSA electrofusion buffer and its cell fusion efficiency, viability and screening of positive wells The electrofusion buffer of different concentrations of BSA of the present invention is configured as follows: Protocol A. 0.5% BSA protocol: 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.5% BSA Step 1: Weigh 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.5% BSA.

[0044] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0045] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0046] Protocol B. 0.1% BSA protocol: 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA Step 1: Weigh 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0047] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0048] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0049] Solution C. 0% BSA solution: 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O Step 1: Weigh 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, and 0.1 mM (CH3COO)2Mg·4H2O. Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0050] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0051] The experimental results and data are shown in Table 3: Table 3 From the above experimental results and data, by comparing the cell fusion rate, number of positive wells, number of cells and viability after fusion in electrofusion buffer prepared with different concentrations of BSA, it was found that the electrofusion buffer prepared with 0.1% BSA had better effect. Because adding BSA can not only provide nutrients for cells, but also maintain appropriate osmotic pressure and protect the cytoplasm, but excessive BSA will affect cell fusion and reduce the fusion rate. Example 4 The present invention provides a method for preparing hybridoma cells. The method is implemented by using an electrofusion buffer, wherein the electrofusion buffer comprises the following components: 100-300 mM sugar, 0.1-0.2 mM (CH3COO)2Ca·H2O, 0.1-0.2 mM (CH3COO)2Mg·4H2O, and 0.05-0.2% BSA.

[0052] The following study will compare the fusion effects of the most preferred electrofusion buffer of the present invention and three commercially available electrofusion buffers (cell fusion efficiency, viability, and screening of positive wells). The most preferred electrofusion buffer configuration of the present invention is as follows: 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0053] The most preferred specific preparation steps of the electrofusion buffer are as follows: Step 1: Weigh 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

[0054] Step 2: Dissolve the ingredients in step 1 in ultrapure water, measure the pH value at 7.0-7.2, and adjust the volume to 1000 mL.

[0055] Step 3: Sterilize the electrofusion buffer by filtering it with a 0.22 μm filter membrane and store it at 4°C until use.

[0056] The most preferred electrofusion buffer of the present invention and three commercially available electrofusion buffers (commodity A, commodity B, commodity C) were applied to the preparation of hybridoma cells, and the cell fusion efficiency, viability and screening of positive wells of the most preferred electrofusion buffer of the present invention and the three commercially available electrofusion buffers were compared. The specific steps are as follows: (1) Under sterile conditions, prepare mouse spleen cell suspension and count the cells. Mix the mouse spleen cells with the SP2 / 0 myeloma cells that have been collected and counted in advance at a ratio of 2:1–5:1. (2) Resuspend the cells in step (1) in the above electrofusion buffer, add 5 mL of electrofusion buffer to wash, centrifuge at 1000 rpm for 3 min, and repeat the operation twice; (3) Prepare the fusion chamber of the electrofusion instrument by rinsing it with electrofusion buffer in advance, and set the fusion parameters in advance, including three AC electric fields and one DC pulse voltage; (4) The first AC electric field allows the cells to begin pre-arrangement. The specific parameters are: initial voltage VO1: 30 V, peak voltage VF1: 50 V, AC electric field frequency 2 MHz, and duration of action is 20 seconds.

[0057] (5) The second AC electric field allows the cells to be evenly arranged. The specific parameters are: initial voltage VO2: 60 V, peak voltage VF2: 60 V, AC electric field frequency 2 MHz, and duration of action is 20 seconds.

[0058] (6) The third DC pulse is the key to cell fusion. The specific parameters are: DC pulse voltage 600 V, action time 50 μs, pulse number 1; (7) The last AC electric field restores the cell state. The specific parameters are: initial voltage VO3: 50 V, peak voltage VF3: 5 V, AC electric field frequency 0.8 MHz, and duration of action 7 seconds; (8) Add the cell suspension from step (2) into the fusion chamber of the electrofusion instrument that has been pre-rinsed with buffer. Be careful not to generate bubbles when adding the cell suspension. Fusion should be performed within 30 s after addition. The cell fusion density should be controlled at 0.5-1×10 7 Pieces / mL.

[0059] (9) performing cell fusion according to the electrofusion procedure of steps (3) to (8) to form a fusion product containing hybridoma cells; (10) After the fusion is completed, let the cells stand in the fusion pool for 2 minutes, then transfer them to the prepared HAT complete culture medium and incubate them in a 37°C incubator for 60 minutes to allow the cells to adapt and recover. (11) The fused cell suspension was added to a 96-well plate containing feeder cells and cultured for 7 days, followed by screening of positive wells.

[0060] Cell fusion efficiency Figure 1 As shown, cell viability and cell number are Figure 2 As shown, the number of positive wells screened is as follows Figure 3 shown.

[0061] The fusion efficiency (‰) is the total number of hybridoma colonies in each well divided by the number of spleen cells, multiplied by 1000; the cell survival rate examines the proportion of live cells to the total number of cells. Figure 1-3It can be seen that the cell fusion rate and the number of positive wells after fusion with the most preferred electrofusion buffer prepared by the present invention are better than those of commercially available A and B type electrofusion buffers, and are equivalent to those of commercially available C type electrofusion buffer. It can be used in subsequent electrofusion projects for hybridoma cell preparation to reduce fusion costs while improving fusion quality.

[0062] The preferred formulation of the present invention is 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O and 0.1% BSA.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All documents mentioned in the present invention are quoted in full in this application as reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention within the spirit and principles of the present invention, and the modifications of these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. An electrofusion buffer for hybridoma cell preparation, characterized in that: The electrofusion buffer contains the following components: 100-300 mM sugar, 0.1-0.2 mM (CH3COO)2Ca·H2O, 0.1-0.2 mM (CH3COO)2Mg·4H2O, and 0.05-0.2% BSA.

2. The electrofusion buffer according to claim 1, characterized in that Contains the following ingredients: 100 mM D-trehalose, 0.1 mM (CH3COO)2Ca·H2O, 0.1 mM (CH3COO)2Mg·4H2O, and 0.1% BSA.

3. The electrofusion buffer according to claim 1, characterized in that The pH value of the electrofusion buffer is 7.0-7.

2.

4. Use of the electrofusion buffer according to any one of claims 1 to 3 in hybridoma technology or monoclonal antibody technology.

5. A method for preparing hybridoma cells, using the electrofusion buffer according to any one of claims 1 to 3, characterized in that: The method for preparing hybridoma cells specifically comprises the following steps: (1) Prepare mouse spleen cell suspension, wash and count, and mix mouse spleen cells with pre-cultured SP2 / 0 myeloma cells in a ratio of 2:1–5:1; (2) Resuspend the cells from step (1) in electrofusion buffer and wash twice by centrifugation using the buffer; (3) adding the cell suspension from step (2) into the fusion chamber of the electrofusion instrument which has been pre-rinsed with buffer; (4) fusing spleen cells with SP2 / 0 myeloma cells according to an electrofusion procedure to form a fusion product containing hybridoma cells; (5) Add the cell suspension after electrofusion to HAT medium and incubate in a 37°C incubator for 60 min to allow the cells to adapt and recover; (6) Add the fused cell suspension to a 96-well plate containing feeder cells for culture and subsequent screening.

6. The method according to claim 5, characterized in that The cell fusion density in step (3) is 0.5-1×10 7 Pieces / mL.

7. The method according to claim 5, characterized in that The electrofusion procedure of step (4) is as follows: (1) applying two AC voltages; and (2) applying a DC pulse and then applying an AC voltage again.

8. The method according to claim 7, characterized in that The AC voltage parameters of the program (1) are: the first AC electric field is: initial voltage VO1: 30 V, peak voltage VF1: 50 V, AC electric field frequency 2 MHz, and continuous action time is 20 seconds; the second AC electric field is: initial voltage VO2: 60 V, peak voltage VF2: 60 V, AC electric field frequency 2 MHz, and continuous action time is 20 seconds.

9. The method according to claim 7, characterized in that: The DC pulse parameters of the procedure (2) are: DC pulse voltage 600 V, action time 50 microseconds, and pulse number 1.

10. The method according to claim 7, characterized in that The AC voltage parameters of the program (2) are: AC electric field initial voltage VO3: 50 V, peak voltage VF3: 5 V, AC electric field frequency 0.8 MHz, and continuous action time of 7 seconds.

Images