An improved transient transfection method for CHO-S cells

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to an improved transient transfection method for CHO-S cells. This transfection method includes the following steps: passage CHO-S cells for 3 days prior to transient transfection, adjusting the cell density to 0.35 × 10⁻⁶ cells / year. 6 Cells / mL were cultured in a shaker; on the day of transient transfection, the CHO-S cell density was adjusted to 4 × 10⁶ cells / mL. 6 -6×10 6 The cells / mL concentration was used to obtain a CHO-S cell suspension. Before transfection, the prepared solution A and solution B were mixed and allowed to stand to obtain a mixed droplet. The mixed droplet was added to the CHO-S cell suspension for culture to obtain recombinant CHO-S cells. The transfection method of the present invention not only has high transfection efficiency, but also obtains higher expression levels of the target antibody in CHO-S cells, providing a new approach for the development of protein biotherapeutic agents.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an improved method for transient transfection of CHO-S cells. Background Technology

[0002] Producing recombinant proteins at the milligram or gram level is a fundamental process for many research teams and pharmaceutical companies in preclinical, biochemical, biophysical, and drug research. Mammalian cell systems are the optimal choice for recombinant protein expression in the biopharmaceutical field due to their ability to optimally express high-molecular-weight protein products, ensure correct folding of complex protein structures, and provide appropriate post-translational modifications for glycoproteins. Traditional mammalian cell expression systems involve constructing stable expression lines that integrate exogenous genes into the host cell chromosome. Constructing stable expression lines for recombinant protein production requires extensive screening after DNA introduction into cells to identify cell lines with high and stable expression levels. This process typically takes several months and requires significant manpower.

[0003] CHO cells (Chinese hamster ovary cells) are widely used as a mammalian protein expression system for expressing recombinant DNA proteins. For example, CHO-S cells, in their accompanying CHO-S expression medium, exhibit non-clumping and non-aggregating characteristics and can grow to very high cell densities, resulting in even higher protein yields through transient transfection. Transient transfection of CHO-S cells allows for rapid, high-yield transient protein production, enabling laboratory development of protein biotherapeutic agents from the initial stage of CHO-expressed proteins throughout the drug development process. With the rapid development of life sciences and biopharmaceuticals, a large number of proteins are used in basic research and drug development, creating an urgent need for a method to obtain recombinant proteins in a short time. Transient gene expression has emerged to address this need. Unlike stable expression systems, the exogenous DNA in transient transfection does not integrate into the host cell DNA, and its number in the cell typically decreases with cell division. Therefore, protein expression can only be maintained for a few days to a dozen days. However, its advantage is that it can produce gene expression products within a short period of time, and it is highly versatile. However, compared to constructing stable expression systems, transient transfection for recombinant protein production suffers from low transfection efficiency and low expression levels, significantly limiting its application. Furthermore, the commonly used transient transfection method for CHO cells has low transfection efficiency, and the expression yield of the target gene in transfected cells is low, failing to meet practical needs. Therefore, improving the efficiency of transient transfection and increasing expression levels has become a pressing issue in routine operations. Summary of the Invention

[0004] In view of the above-mentioned defects or improvement needs of the existing technology, the present invention provides an improved transient transfection method for CHO-S cells. This transfection method not only has high transfection efficiency, but also can obtain higher expression levels of target antibodies in CHO-S cells, providing new ideas for the development of protein biotherapeutic agents.

[0005] To achieve the above objectives, the present invention provides an improved method for transient transfection of CHO-S cells, comprising the following steps: (1) Three days before transient transfection, CHO-S cells were passaged and cultured, and the cell density was adjusted to 0.35 × 10⁶ cells / year. 6 cells / mL, place a shake flask containing a certain volume of cells into a shaker for culture; (2) On the day of transient transfection, CHO-S cells were counted and diluted with freshly prepared transfection medium to adjust the cell density to 4 × 10⁻⁶. 6 -6×10 6 The cell count was measured at cells / mL, and the cell viability was found to be above 97%, resulting in a CHO-S cell suspension. (3) Before transfection, mix the prepared solution A and solution B and let them stand for a period of time to obtain a mixed droplet; (4) Add the mixed droplets described in step (3) to the CHO-S cell suspension and culture them according to the cell culture conditions to obtain recombinant CHO-S cells.

[0006] Furthermore, in step (1) of the above technical solution, the passage medium is Mirus CHOgro Expression Medium with 4 mmol / L glutamine added; the cell passage culture conditions are a temperature of 37°C, a CO2 concentration of 8%, and a shaking speed of 125 rpm.

[0007] Furthermore, in step (2) of the above technical solution, the transfection medium is 90% Mirus Chogro Expression Medium supplemented with 4 mmol / L glutamine and 10% Gibco. ® A mixture of FreeStyle™ CHO ExpressionMedium.

[0008] Further, in step (3) of the above technical solution, solution A is a solution of plasmid containing antibody light and heavy chains dissolved in NaCl buffer, and solution B is a solution of PEI transfection reagent dissolved in NaCl buffer; the mass ratio of plasmid to PEI transfection reagent in the mixed droplet is 1:3; the plasmid containing antibody light and heavy chains is a mixture of plasmid containing antibody light chains and plasmid containing antibody heavy chains in a mass ratio of 6:4; the standing temperature is 25-37℃, and the time is 5-20min.

[0009] Furthermore, in the above technical solution, the ratio of the total mass of the antibody light and heavy chain plasmids to the number of cells in the CHO-S cell suspension is 0.5-1 μg: 1 × 10⁻⁶. 6 cells.

[0010] Furthermore, in step (4) of the above technical solution, the concentration of total plasmid in the CHO-S cell suspension is 3-4 μg / mL; the cell culture conditions are: cultured on a shaker at a temperature of 37°C, a carbon dioxide concentration of 8%, a shaker speed of 125 rpm, and a transfection time of 48-72 h.

[0011] The present invention also provides a recombinant CHO-S cell prepared by the above-described modified CHO-S cell transient transfection method.

[0012] The present invention also provides a method for promoting antibody expression after transient transfection of CHO-S cells. The recombinant CHO-S cells obtained after transient transfection are cultured with sodium butyrate solution and EN3 solution for 18-20 hours after transfection to promote protein expression in the transfected cells.

[0013] Furthermore, in the above technical solution, the sodium butyrate solution is a sterile solution with a concentration of 100mM using PBS buffer as a solvent, and its addition amount is 25μL per milliliter of transfection solution; the EN3 solution is a sterile solution after dissolving 29.2g of glutamine and 200g of glucose in each L of serum-free 1640 culture medium, and its addition amount is 50μL per milliliter of transfection solution.

[0014] The present invention also provides the application of the modified CHO-S cell transient transfection method described above for the preparation of recombinant CHO-S cells in recombinant protein expression.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves high transfection efficiency and yields higher expression levels of the target protein in CHO-S cells by controlling the number of CHO-S cell suspensions, optimizing the transfection reagent ratio, transfection time, and transfection culture medium components. Simultaneously, the addition of a certain amount of hydrolysate at a specific time after transfection promotes antibody expression in transfected cells. Compared to traditional transfection methods, this approach achieves significantly higher expression levels of the target protein in CHO-S cells, providing a new approach for the development of protein biotherapeutic agents. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 These are microscopic images of the state of CHO-S cells after passage culture according to the present invention. Figure 2 This is a comparison chart of transfection efficiency in Examples 1-3 of the present invention; Figure 3 This is a comparison chart of the transfection efficiency of Comparative Examples 1-2 and Example 3 of the present invention; Figure 4 This is a comparison chart of the transfection efficiency of Comparative Examples 3-4 and Example 3 of the present invention; Figure 5 This is a comparison chart of the transfection efficiency of Comparative Examples 5-6 and Example 3 of the present invention; Figure 6 This is a comparison chart of the transfection efficiency of Comparative Examples 7-8 and Example 3 of the present invention. Detailed Implementation

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials used in the following examples are all commercially available products and can be purchased from the market.

[0019] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0020] The raw materials involved in the various embodiments of this invention are either commercially available products or can be prepared according to existing methods. The antibody heavy and light chain plasmids, which contain expression plasmids containing antibody heavy and light chains respectively, were purchased from Excellgene.

[0021] This invention discloses a transient transfection method for CHO-S cells, comprising the following steps: (1) Three days before transient transfection, CHO-S cells were passaged and cultured, and the cell density was adjusted to 0.35 × 10⁶ cells / year. 6 cells / mL, the shake flask containing a certain volume of cells is placed in a shaker for culture; specifically, the shake flask is a round-bottom shake flask of 250mL or 500mL for passage; the certain volume is either 30mL or 60mL.

[0022] (2) On the day of transient transfection, CHO-S cells were counted and diluted with freshly prepared transfection medium to adjust the cell density to 4 × 10⁻⁶. 6 -6×10 6 The cell count was measured at cells / mL, and the cell viability was found to be above 97%, resulting in a CHO-S cell suspension.

[0023] (3) Before transfection, mix the prepared solution A and solution B and let them stand for a period of time to obtain a mixed droplet. Specifically, the standing method is to keep the room temperature at no less than 25°C and let it stand for 5-20 minutes.

[0024] (4) Add the mixed droplets described in step (3) to the CHO-S cell suspension and culture according to the cell culture conditions to obtain recombinant CHO-S cells. Specifically, a 125mL round-bottom shake flask is used for transfection.

[0025] Specifically, the mass ratio of the plasmid containing the antibody light and heavy chains to the PEI transfection reagent in the mixed droplet is 1:3; Solution A is a solution containing antibody light and heavy chains in NaCl buffer, and solution B is a solution containing PEI transfection reagent in NaCl buffer; the mass ratio of antibody light chain plasmid to antibody heavy chain plasmid in the antibody light and heavy chain plasmid is 6:4.

[0026] More specifically, in a 60 mL transfection system, the mixed droplet contains solution A, which is prepared by mixing plasmid containing antibody light and heavy chains with NaCl buffer at a ratio of 240 μg: 1 mL, and solution B, which is prepared by mixing PEI transfection reagent with NaCl buffer at a ratio of 720 μg: 1 mL.

[0027] Specifically, the concentration of total plasmids in the CHO-S cell suspension is 3-4 μg / mL, and more specifically, the ratio of total plasmid amount to the number of cells in the CHO-S cell suspension is 0.5-1 μg: 1 × 10⁻⁶. 6 Cells / mL.

[0028] As a preferred technical solution, the CHO-S cells should be passaged once within 24 hours of transfection. Cell passage culture is performed using Mirus CHOgro Expression Medium supplemented with 4 mmol / L glutamine, under the following conditions: 37°C, 8% CO2, and a shaking speed of 125 rpm. As a preferred technical solution, the newly prepared transfection medium is a serum-free medium containing 90% Mirus CHOgro Expression Medium and 10% Gibco® FreeStyle™ CHO Expression Medium with a final concentration of 4 mmol / L glutamine; the transfection time is 48-72 h.

[0029] This invention discloses a method for promoting antibody expression in CHO-S cells after transient transfection, comprising the following steps: Recombinant CHO-S cells obtained after transient transfection are added with 25 μL sodium butyrate solution / mL transfection volume and 50 μL LEN3 solution / mL transfection volume 18-20 h after transfection to promote protein expression in the transfected cells; the cell culture conditions after transfection are 37℃, 8% CO2, and a shaking speed of 125 rpm.

[0030] Specifically, the sodium butyrate solution is a 100mM sterile solution using PBS buffer as the solvent, and the EN3 solution is a sterile solution containing 29.2g of glutamine and 200g of glucose per 1L of serum-free 1640 culture medium.

[0031] The following specific examples will provide further details.

[0032] Example 1 A transient transfection method for CHO-S cells includes the following steps: (1) Take CHO-S cells with good growth and passage them 3 days before transient transfection. Then, use Mirus CHOgro Expression Medium supplemented with 4 mmol / L glutamine to adjust the cell density to 0.35 × 10⁶ cells / year. 6 The CHO-S cell suspension was prepared at cells / mL and placed in a 250mL Erlenmeyer flask for culture on a shaker. The shaker settings were: temperature 37℃, carbon dioxide concentration 8%, and shaker speed 125rpm. The state of the CHO-S cells after passaged culture was as follows. Figure 1 As shown; (2) On the day of transient transfection, the CHO-S cell slurry obtained in step (1) was used for counting and observation of cell status. The cell density was adjusted to 4×10⁶ cells by diluting the cells with freshly prepared transfection medium (90% Mirus CHOgro Expression Medium and 10% Gibco® FreeStyle™ CHOExpression Medium + 4 mmol / L glutamine). 6 The cell count / mL was such that the cell viability was above 97%, thus obtaining the CHO-S cell suspension. (3) Before transfection, mix the prepared solution A and solution B, place them at room temperature, and let them stand for 5 minutes to equilibrate, thus obtaining a mixed droplet; specifically, the preparation method of the mixed droplet used in the 60 mL transfection system is as follows: ① A 240 μg antibody plasmid mixture was prepared by combining 144 μg of antibody light chain plasmid and 96 μg of antibody heavy chain plasmid. ②Prepare solution A: Add 1 mL of NaCl buffer to the total plasmid tube; ③ Prepare solution B: Add 720 μg of PEI to 1 mL of NaCl buffer for equilibration; ④ Let the above solutions A and B stand at room temperature for 10 minutes to equilibrate; ⑤ Slowly add the balanced solution A to solution B, and let the AB mixture stand at room temperature for 5 minutes to equilibrate, thus obtaining the mixed droplet; (4) Add the mixed droplets described in step (3) into a 125 mL round-bottom shake flask containing CHO-S cell suspension (where the total plasmid concentration in the transfection system is 4 μg / mL and the PEI concentration is 12 μg / mL), place it in a shaker for culture, and set the culture parameters as follows: temperature 37℃, carbon dioxide concentration 8%, shaker speed 125 rpm, transfect for 48 h to obtain recombinant CHO-S cells.

[0033] Example 2 A transient transfection method for CHO-S cells includes the following steps: (1) Take CHO-S cells with good growth and passage them 3 days before transient transfection. Then, use Mirus CHOgro Expression Medium supplemented with 4 mmol / L glutamine to adjust the cell density to 0.35 × 10⁶ cells / year. 6 The cells / mL were placed in a 250mL Erlenmeyer flask and cultured in a shaker. The shaker was set with the following parameters: temperature 37℃, carbon dioxide concentration 8%, and shaking speed 125rpm. (2) On the day of transient transfection, the CHO-S cell slurry obtained in step (1) was used for counting and observation of cell status. The cell density was adjusted to 5×10⁶ cells by diluting the cells with freshly prepared transfection medium (90% Mirus CHOgro Expression Medium and 10% Gibco® FreeStyle™ CHOExpression Medium + 4 mmol / L glutamine). 6 The cell count / mL was such that the cell viability was above 97%, thus obtaining the CHO-S cell suspension. (3) Before transfection, mix the prepared solution A and solution B, place them at room temperature, and let them stand for 5 minutes to equilibrate, thus obtaining a mixed droplet; specifically, the preparation method of the mixed droplet used in the 60 mL transfection system is as follows: ① A 180 μg antibody plasmid mixture was prepared by combining 108 μg of antibody light chain plasmid and 72 μg of antibody heavy chain plasmid. ②Prepare solution A: Add 1 mL of NaCl buffer to the total plasmid tube; ③ Prepare solution B: Add 540 μg of PEI to 1 mL of NaCl buffer for equilibration; ④ Let the above solutions A and B stand at room temperature for 10 minutes to equilibrate; ⑤ Slowly add the balanced solution A to solution B, and let the AB mixture stand at room temperature for 15 minutes to obtain the mixed droplet; (4) Add the mixed droplets described in step (3) into a 125 mL round-bottom shake flask containing CHO-S cell suspension (where the total plasmid concentration in the transfection system is 3 μg / mL and the PEI concentration is 9 μg / mL), place it in a shaker for culture, and set the culture parameters as follows: temperature 37℃, carbon dioxide concentration 8%, shaker speed 125 rpm, transfect for 72 h to obtain recombinant CHO-S cells.

[0034] Example 3 A transient transfection method for CHO-S cells includes the following steps: (1) Take CHO-S cells with good growth and passage them 3 days before transient transfection. Then, use Mirus CHOgro Expression Medium supplemented with 4 mmol / L glutamine to adjust the cell density to 0.35 × 10⁶ cells / year. 6 The cells / mL were placed in a 250mL Erlenmeyer flask and cultured in a shaker. The shaker was set with the following parameters: temperature 37℃, carbon dioxide concentration 8%, and shaking speed 125rpm. (2) On the day of transient transfection, the CHO-S cell slurry obtained in step (1) was used for counting and observation of cell status. The cell density was adjusted to 6×10⁶ cells by diluting the cells with freshly prepared transfection medium (90% Mirus CHOgro Expression Medium and 10% Gibco® FreeStyle™ CHOExpression Medium + 4 mmol / L glutamine). 6 The cell count / mL was such that the cell viability was above 97%, thus obtaining the CHO-S cell suspension. (3) Before transfection, mix the prepared solution A and solution B, place them at room temperature, and let them stand for 5 minutes to equilibrate, thus obtaining a mixed droplet; specifically, the preparation method of the mixed droplet used in the 60 mL transfection system is as follows: ① A 240 μg antibody plasmid mixture was prepared by combining 144 μg of antibody light chain plasmid and 96 μg of antibody heavy chain plasmid. ②Prepare solution A: Add 1 mL of NaCl buffer to the total plasmid tube; ③ Prepare solution B: Add 720 μg of PEI to 1 mL of NaCl buffer for equilibration; ④ Let the above solutions A and B stand at room temperature for 10 minutes to equilibrate; ⑤ Slowly add the balanced solution A to solution B, and let the AB mixture stand at room temperature for 20 minutes to obtain the mixed droplet; (4) Add the mixed droplets described in step (3) into a 125 mL round-bottom shake flask containing CHO-S cell suspension (where the total plasmid concentration in the transfection system is 4 μg / mL and the PEI concentration is 12 μg / mL), place it in a shaker for culture, and set the culture parameters as follows: temperature 37℃, carbon dioxide concentration 8%, shaker speed 125 rpm, transfect for 72 h to obtain recombinant CHO-S cells.

[0035] Comparative Example 1 The difference from Example 3 is that in step (2), the cell density is 8 × 10⁻⁶. 6 cells / mL.

[0036] Comparative Example 2 The difference from Example 3 is that in step (2), the cell density is 3×10⁻⁶. 6 cells / mL.

[0037] Comparative Example 3 The difference from Example 3 is that in step (3), the preparation method of the mixed drops used in the 60 mL transfection system is as follows: ① A 120 μg antibody plasmid mixture was prepared by combining 72 μg of antibody light chain plasmid and 48 μg of antibody heavy chain plasmid. ②Prepare solution A: Add 1 mL of NaCl buffer to the total plasmid tube; ③ Prepare solution B: Add 360 μg of PEI to 1 mL of NaCl buffer for equilibration; ④ Let the above solutions A and B stand at room temperature for 10 minutes to equilibrate; ⑤ Slowly add the balanced solution A to solution B, and let the AB mixture stand at room temperature for 20 minutes to obtain the mixed droplet.

[0038] When added to the transfection system, the total plasmid concentration was 2 μg / mL and the PEI concentration was 6 μg / mL. Comparative Example 4 The difference from Example 3 is that in step (4), the transfection time is 24 hours.

[0039] Comparative Example 5 The difference from Example 3 is that in step (2), the culture medium used is Mirus CHOgro Expression Medium + 4 mmol / L glutamine.

[0040] Comparative Example 6 The difference from Example 3 is that in step (2), the culture medium used is 80% Mirus CHOgro Expression Medium and 20% Gibco® FreeStyle™ CHO Expression Medium + 4 mmol / L glutamine.

[0041] Comparative Example 7 The difference from Example 3 is that in step (3), the preparation method of the mixed drops used in the 60 mL transfection system is as follows: ① A 240 μg antibody plasmid mixture was prepared by combining 144 μg of antibody light chain plasmid and 96 μg of antibody heavy chain plasmid. ②Prepare solution A: Add 1 mL of NaCl buffer to the total plasmid tube; ③ Prepare solution B: Add 960 μg of PEI to 1 mL of NaCl buffer for equilibration; ④ Let the above solutions A and B stand at room temperature for 10 minutes to equilibrate; ⑤ Slowly add the balanced solution A to solution B, and let the AB mixture stand at room temperature for 20 minutes to obtain the mixed droplet.

[0042] When added to the transfection system, the total plasmid concentration was 4 μg / mL and the PEI concentration was 16 μg / mL. Comparative Example 8 A transient transfection method for CHO-S cells is summarized below: (1) Take well-grown CHO-S cells with a cell viability of over 95% and adjust the cell density to 5 × 10⁶ cells / year using fresh Dynamis medium. 6 10 cells / mL, add 30mL of culture volume to a 125mL multi-cell shake flask, and temporarily place it in a shaker for culture. The culture parameters are: temperature 37℃, carbon dioxide concentration 8%, and shaker speed 125rpm. (2) Prepare the transfection system during this period. The preparation method is as follows: ① A 240 μg antibody plasmid mixture was prepared by combining 144 μg of antibody light chain plasmid and 96 μg of antibody heavy chain plasmid. ②Prepare solution A: Add 1 mL of NaCl buffer to the total plasmid tube; ③ Prepare solution B: Add 720 μg of PEI to 1 mL of NaCl buffer for equilibration; ④ Let the above solutions A and B stand at room temperature for 10 minutes to equilibrate; ⑤ Slowly add the balanced solution A to solution B, and let the AB mixture stand at room temperature for 20 minutes to equilibrate. (3) Slowly drip the balanced AB solution into the above-mentioned CHO-S cell suspension to be transfected, and place it in a shaker for culture. The culture parameters are: temperature 37℃, carbon dioxide concentration 8%, and shaker speed 125rpm. (4) Sample and test cell growth every day. After 72 hours of transfection, recombinant CHO-S cells are obtained when the cell viability is less than 97%.

[0043] Test case 1. The transfection efficiency and cell viability of the recombinant CHO-S cells obtained in Examples 1-3 and Comparative Examples 1-8 were detected, and the results are shown in Table 1.

[0044] Table 1

[0045] 2. The expression levels of recombinant CHO-S cells obtained in Examples 1-3 and Comparative Examples 1-8 were tested using the following methods, and the results are as follows: Figure 2-6 As shown in the figure. The expression method was as follows: 18-20 h after transfection, the obtained recombinant CHO-S cells were treated with 25 μL sodium butyrate solution / mL transfection volume and 50 μL EN3 solution / mL transfection volume to promote protein expression in the transfected cells. The culture conditions after cell transfection were 37℃, 8% CO2, and a shaking speed of 125 rpm. The sodium butyrate solution was a 100 mM sterile solution using PBS buffer as the solvent, and the EN3 solution was a sterile solution containing 29.2 g glutamine and 200 g glucose per 1 L of serum-free 1640 medium.

[0046] From Table 1 and Figure 2-6 The results show that the recombinant CHO-S cells obtained by the transfection method of the present invention not only have a high transfection rate, but also a high cell survival rate. The expression level of the target protein in CHO-S cells is also significantly higher than that in each control group, indicating that the transfection method of the present invention can obtain a higher expression level of the target protein in CHO-S cells, which can provide a new idea for the development of protein biotherapy agents.

[0047] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An improved method for transient transfection of CHO-S cells, characterized in that, Includes the following steps: (1) Three days before transient transfection, CHO-S cells were passaged and cultured, and the cell density was adjusted to 0.35 × 10⁶ cells / year. 6 cells / mL, the shake flask containing a certain volume of cells was placed in a shaker for culture; the passage medium was Mirus CHOgro Expression Medium supplemented with 4 mmol / L glutamine; (2) On the day of transient transfection, CHO-S cells were counted and diluted with freshly prepared transfection medium to adjust the cell density to 4 × 10⁻⁶. 6 -6×10 6 Cells / mL were measured, and cell viability was found to be above 97%, resulting in a CHO-S cell suspension. The transfection medium consisted of 90% Mirus CHOgro Expression Medium supplemented with 4 mmol / L glutamine and 10% Gibco. ® A mixture of FreeStyle™ CHO Expression Medium; (3) Before transfection, mix the prepared solution A and solution B and let them stand for a period of time to obtain a mixed droplet; solution A is a solution of plasmid containing antibody light and heavy chains dissolved in NaCl buffer, and solution B is a solution of PEI transfection reagent dissolved in NaCl buffer; the mass ratio of plasmid to PEI transfection reagent in the mixed droplet is 1:3; the plasmid containing antibody light and heavy chains is a mixture of plasmid containing antibody light chain and plasmid containing antibody heavy chain in a mass ratio of 6:4; (4) Add the mixed droplets described in step (3) to the CHO-S cell suspension and culture them according to the cell culture conditions to obtain recombinant CHO-S cells.

2. The improved CHO-S cell transient transfection method according to claim 1, characterized in that, In step (1), the cell passage culture conditions are 37°C, CO2 concentration 8%, and shaking speed 125 rpm.

3. The improved CHO-S cell transient transfection method according to claim 1, characterized in that, In step (3), the settling temperature is 25-37℃ and the time is 5-20min.

4. The improved CHO-S cell transient transfection method according to claim 1, characterized in that, The ratio of the total mass of the plasmid to the number of cells in the CHO-S cell suspension is 0.5-1 μg: 1 × 10⁻⁶. 6 cells.

5. The improved CHO-S cell transient transfection method according to claim 1, characterized in that, In step (4), the concentration of total plasmid in the CHO-S cell suspension is 3-4 μg / mL; the cell culture conditions are: cultured on a shaker at a temperature of 37°C, a carbon dioxide concentration of 8%, a shaker speed of 125 rpm, and a transfection time of 48-72 h.

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