A culture medium, method and application suitable for high-density transient transfection of CHO cells

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a culture medium, method and application suitable for high-density transient transfection of CHO cells. The culture medium includes a basal culture medium, a feed culture medium and an intermediate additive, wherein the basal culture medium is a CHO serum-free basal culture medium CE06, and the feed culture medium is a CHO serum-free transient feed culture medium CA06. The protein yield of the process of the present application can exceed 1.85 g / L on the 10th day after transfection, an increase of more than 60% year-on-year. For the CHO transient transfection system, especially for difficult-to-express proteins, the improvement is very significant.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a culture medium, a method and an application suitable for high-density transient transfection of CHO cells. Background Art

[0002] Transient gene expression (TGE) technology has become an important tool for early exploration and large-scale high-throughput protein screening in the research and development stage due to its rapid generation of recombinant proteins, short cycle, simple operation, and high cost-effectiveness. In mammalian cell expression systems, CHO and HEK293 have been widely used in the production of various difficult-to-express or special-need proteins, especially CHO cells, which have post-translational modifications similar to humans and secrete fewer endogenous proteins. Therefore, although the protein expression level of CHO transient transfection is generally lower than that of HEK 293, in CHO transient transfection, it is necessary to ensure a high yield, and the cost also limits the development of the industry. Both liposome transfection and electroporation are too expensive, such as Gibco's transfection kit, but due to the high price, the liposome transfection method can only be used at the laboratory level and cannot be industrialized. Therefore, using PEI-Max transfection is more cost-effective. If its protein yield can be increased, there will be a significant price advantage.

[0003] During CHO transient transfection, transfection efficiency and obtained protein products are affected by many factors, including vector type, culture medium selection, cell density, transfection reagent type, and the ratio of PEI-Max to DNA. After years of efforts by R&D personnel, due to the toxicity of PEI-Max and plasmid restrictions, it is difficult to further improve the optimization of the above conventional factors, especially for the expression proteins used in CHO transient transfection, which are diverse and difficult to express. Usually, in order to improve the transfection efficiency, sodium butyrate, sodium valproate and other substances are added, but they are essentially more toxic to cells and reduce cell growth density. Therefore, how to improve the efficiency of transient transfection of CHO cells is a hot spot and pain point in industry research. Summary of the invention

[0004] The object of the present invention is to provide a culture medium for high-density transient transfection of CHO cells, which can support high-density cell culture.

[0005] Another object of the present invention is to provide a high-density transient transfection method, which can obtain more expression products with a smaller transfection system, increase the expression amount, and thus enhance its application in industrial customers.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A culture medium suitable for high-density transient transfection of CHO cells, comprising a basal culture medium, a feed culture medium and an intermediate additive, wherein the basal culture medium is a CHO serum-free basal culture medium CE06, and the feed culture medium is a CHO serum-free transient transfection feed culture medium CA06;

[0008] The intermediate additives include: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L -Alanine 5g / L, L-glutamine 4g / L, adenosine 0.02~0.05g / L, uridine 0.02~0.05g / L, guanosine 0.02~0.05g / L, any one of sodium butyrate 0.2g / L or sodium propionate 0.2g / L or valproic acid 0.2~0.8g / L, insulin growth factor 0.0004~0.0008g / L, manganese sulfate 0.000002~0.000008g / L, calcium chloride 0.01~0.02g / L, magnesium chloride 0.5~1g / L, zinc sulfate 0.01~0.04g / L.

[0009] Preferably, the intermediate additives include: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamine Acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02~0.05g / L, uridine 0.02~0.05g / L, guanosine 0.02~0.05g / L, any one of sodium propionate 0.2g / L or valproic acid 0.2~0.8g / L, insulin growth factor 0.0004~0.0008g / L, manganese sulfate 0.000005~0.000008g / L, calcium chloride 0.01~0.02g / L, magnesium chloride 0.5~1g / L, zinc sulfate 0.01~0.04g / L.

[0010] Preferably, the intermediate additives include: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 1 0g / L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, valproic acid 0.2g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.01g / L, magnesium chloride 0.5g / L, zinc sulfate 0.04g / L.

[0011] Preferably, the CHO cells are Expi CHO-S cells.

[0012] An application of a culture medium suitable for high-density transient transfection of CHO cells, comprising any of the above-mentioned culture medium suitable for high-density transient transfection of CHO cells, used for culturing CHO cells.

[0013] A culture method suitable for high-density transient transfection of CHO cells, using any of the culture media suitable for high-density transient transfection of CHO cells as described above, specifically comprising the following steps:

[0014] Step 1: CHO cells were cultured in CHO serum-free transient basal medium CE06 at a cell density of 0.2 × 10 6 The number of cells / mL was subcultured every three days. After three subcultures, it was used for transfection experiments to ensure that the cell viability was greater than 90%;

[0015] Step 2: On the day of transfection, 10~20×10 6 The cell number / mL inoculation density was calculated, the required seed cell solution was calculated, the supernatant was removed by centrifugation, fresh CHO serum-free transient basal medium CE06 medium was added to prepare a cell suspension, PEI-Max / DNA complex was added, the PEI-Max concentration in the PEI-Max / DNA complex was 2-4 mg / L, the DNA concentration in the PEI-Max / DNA complex was 6-20 mg / L, and the cells were placed in a shaker for culture under the following culture conditions: 37°C, 5% carbon dioxide, 100-120 rpm;

[0016] Step 3: 4 hours after transfection, add CHO serum-free transient feed medium CA06 at a volume ratio of 5%.

[0017] After culturing for 24 hours, the intermediate additive was added to supplement sugar to 12 g / L, and the culture temperature was lowered from 37°C to 33°C, and the culture was continued.

[0018] After 48 hours of culture, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%, and sugar was supplemented to 12 g / L.

[0019] After 72 hours of culture, sugar was added to 12 g / L.

[0020] After 96 hours of culture, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%, and sugar was supplemented to 12 g / L.

[0021] The sugar content was monitored daily and replenished to 12 g / L in time. The cells were harvested on the 10th day of culture.

[0022] Preferably, in step 2, on the day of transfection, the inoculation density is 10×10 6 The number of cells / mL, the mass ratio of PEI-Max to DNA in the PEI-Max / DNA complex was 1:4.

[0023] The beneficial effects of the present invention are mainly that the protein yield of the process of the present application can exceed 1.85 g / L on the 10th day after transfection, an increase of more than 60% year-on-year. For the CHO transient transfection system, especially for difficult-to-express proteins, the improvement is very significant.

[0024] The mechanism of action of the present invention is mainly as follows: the present invention first develops a high-density transfection process through DoE experiments, matches CHO serum-free transient transfection basal medium CE06 and CHO serum-free transient transfection feed medium CA06, and by adding intermediate additives (amino acids and nucleosides and insulin growth factor, metal elements), increases the density of living cells, maintains a high viability, and thus increases the protein expression amount.

[0025] The guanosine contained in the present invention is a substrate for synthesizing DNA and RNA, and is very important in cell signal transduction, thereby affecting metabolic pathways and increasing protein expression. Insulin growth factor can regulate the cell's uptake, utilization and storage of glucose, amino acids and fatty acids, promote cell growth, increase the density of living cells, and increase protein expression. Zinc sulfate is an antioxidant that can effectively resist reactive oxygen, stabilize and protect cells, and a certain concentration can inhibit cell apoptosis, thereby increasing protein expression. Manganese sulfate can prevent tyrosine from being oxidized, and tyrosine can prolong the survival time of cells, thereby further increasing protein expression. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Required commercial culture media: basal culture medium (CHO serum-free transient transfection basal culture medium CE06, catalog number: CE000-N052, brand: Suzhou Ecosine Biotechnology Co., Ltd.), feed medium (CHO serum-free transient transfection feed medium CA06, catalog number: CA000-N031, brand: Suzhou Ecosine Biotechnology Co., Ltd.), Expi CHO-S cells (catalog number: A37785, brand: Gibco), plasmid containing adalimumab gene (constructed by Suzhou Genewise Biotechnology Co., Ltd.), PEI-Max (catalog number: 24765 (100), brand: Polysciences).

[0028] Cell transfection operation process:

[0029] Prepare Expi CHO-S cells and culture them in CHO serum-free transient basal medium CE06 at a cell density of 0.2 × 10 6 The number of cells / mL was subcultured every three days. After three subcultures, it was used for transfection experiments to ensure that the cell viability was greater than 90%;

[0030] Further, according to Table 1, the DoE design was conducted;

[0031] Table 1

[0032]

[0033] On the day of transfection in the following process, refer to Table 1 for the inoculation density and the concentration of PEI-Max / DNA complex.

[0034] Furthermore, on the day of transfection, the required seed cell solution was calculated according to the inoculation density, the supernatant was removed by centrifugation, and 18 mL of fresh CHO serum-free transient basal medium CE06 medium was added and placed in a 125 mL shake flask;

[0035] Further, PEI-Max was diluted to 1 mL using CHO serum-free transient basal medium CE06 and incubated at room temperature for 5 min.

[0036] Further, the DNA was diluted to a 1 mL system using CHO serum-free transient basal medium CE06;

[0037] Further, PEI-Max was added to DNA to form a PEI-Max / DNA complex, mixed well, and incubated at room temperature for 10 min;

[0038] Further, the prepared 2 mL PEI-Max / DNA complex was slowly added to the cell suspension containing 18 mL fresh CHO serum-free transient basal medium CE06. In this step, the mixture diluent was added while shaking the shake flask to ensure that the cells and the mixture were fully contacted and mixed. The culture system was 20 mL in total and placed in a carbon dioxide shaker for culture (culture conditions: 37°C, 5% carbon dioxide, 100-120 rpm);

[0039] Furthermore, 4 h after transfection, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%;

[0040] Further, after culturing for 24 hours, the intermediate additive (Example 1-8) was added, and no intermediate additive was added as a control group, sugar was supplemented to 12 g / L, the temperature of the carbon dioxide incubator was reduced from 37° C. to 33° C., and the culture was continued;

[0041] Furthermore, after 48 hours of culture, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%, and sugar was supplemented to 12 g / L;

[0042] Furthermore, after 72 h of culture, sugar was supplemented to 12 g / L;

[0043] Furthermore, after 96 hours of culture, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%, and sugar was supplemented to 12 g / L;

[0044] Furthermore, the sugar content was monitored every day and the sugar was supplemented to 12 g / L in time. On the 10th day of culture, the cells were harvested, the cell supernatant was taken, and the expression level of adalimumab protein was detected by high performance liquid chromatography.

[0045] Furthermore, through DoE screening, the best process was determined to be: Experimental Group 5, Examples 1-8 all use this process.

[0046] Example 1: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, sodium butyrate 0.2g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.02g / L, magnesium chloride 1g / L, zinc sulfate 0.02g / L;

[0047] Example 2: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.05g / L, uridine 0.05g / L, guanosine 0.05g / L, sodium propionate 0.2g / L, insulin growth factor 0.0004g / L, manganese sulfate 0.000005g / L, calcium chloride 0.02g / L, magnesium chloride 0.5g / L, zinc sulfate 0.01g / L;

[0048] Example 3: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, valproic acid 0.4g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.02g / L, magnesium chloride 0.5g / L, zinc sulfate 0.02g / L;

[0049] Example 4: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.04g / L, uridine 0.04g / L, guanosine 0.04g / L, valproic acid 0.4g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.02g / L, magnesium chloride 0.8g / L, zinc sulfate 0.02g / L;

[0050] Example 5: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, valproic acid 0.4g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000002g / L, calcium chloride 0.02g / L, magnesium chloride 0.5g / L, zinc sulfate 0.04g / L;

[0051] Example 6: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, valproic acid 0.8g / L, insulin growth factor 0.0004g / L, manganese sulfate 0.000008g / L, calcium chloride 0.02g / L, magnesium chloride 1g / L, zinc sulfate 0.01g / L;

[0052] Example 7: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L, L-valine Amine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.04g / L, uridine 0.04g / L, guanosine 0.04g / L, valproic acid 0.4g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.02g / L, magnesium chloride 0.5g / L, zinc sulfate 0.04g / L, dimethyl sulfoxide 5mL / L;

[0053] Example 8: Specifically comprising: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamate 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, valproic acid 0.2g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.01g / L, magnesium chloride 0.5g / L, zinc sulfate 0.04g / L.

[0054] In the table, D00 (day 0), D02 (day 2), D04 (day 4), D06 (day 6), D08 (day 8), and D10 (day 10).

[0055] Table 2: Viable cell density

[0056]

[0057] Table 3: Cell viability

[0058]

[0059] Table 4: Protein expression

[0060]

[0061] Tables 2 to 4 show that the viable cell density of the high-density transfected group was higher than that of the conventional experimental group, and the cell viability was also well maintained, with the highest expression level reaching twice that of the conventional experimental group.

[0062] Table 5: Viable cell density

[0063]

[0064] Table 6: Cell viability

[0065]

[0066] Table 7: Protein expression

[0067]

[0068] Tables 5 to 7 show that, based on experimental group 5, additional intermediate additives were added, and the protein expression of Example 8 was better than that of other examples and significantly higher than that of the control group. The protein yield of this process on the 10th day after transfection can exceed 1.85 g / L, an increase of more than 60% year-on-year. For CHO transient transfection system, especially for difficult-to-express proteins, this improvement is very significant.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A culture method suitable for high-density transient transfection of CHO cells, characterized in that: The culture medium suitable for high-density transient transfection of CHO cells includes a basal culture medium, a feed culture medium and an intermediate additive, wherein the basal culture medium is a CHO serum-free basal culture medium CE06, and the feed culture medium is a CHO serum-free transient transfection feed culture medium CA06; The intermediate additives are: L-aspartic acid 4g / L, L-serine 1.5g / L, L-glycine 1.2g / L, L-asparagine 2g / L, L-arginine 3g / L, L-histidine 2g / L, L-isoleucine 4g / L, L-leucine 12g / L, L-lysine 3g / L, L-methionine 2g / L, L-phenylalanine 5g / L, L-threonine 4g / L, L-tryptophan 2.8g / L, L-tyrosine 10g / L L, L-valine 3g / L, L-cysteine ​​4.4g / L, L-glutamic acid 4g / L, L-alanine 5g / L, L-glutamine 4g / L, adenosine 0.02g / L, uridine 0.02g / L, guanosine 0.02g / L, valproic acid 0.2g / L, insulin growth factor 0.0008g / L, manganese sulfate 0.000005g / L, calcium chloride 0.01g / L, magnesium chloride 0.5g / L, zinc sulfate 0.04g / L; The specific steps include: Step 1: CHO cells were cultured in CHO serum-free transient basal medium CE06 at a cell density of 0.2 × 10 6 The number of cells / mL was subcultured every three days. After three subcultures, it was used for transfection experiments to ensure that the cell viability was greater than 90%; Step 2: On the day of transfection, 10 × 10 6 The cell number / mL inoculation density was calculated, the required seed cell solution was calculated, the supernatant was removed by centrifugation, fresh CHO serum-free transient basal medium CE06 medium was added to prepare a cell suspension, PEI-Max / DNA complex was added, the PEI-Max concentration in the PEI-Max / DNA complex was 3 mg / L, the DNA concentration in the PEI-Max / DNA complex was 12 mg / L, and the cells were placed in a shaker for culture under the following culture conditions: 37°C, 5% carbon dioxide, 100-120 rpm; Step 3: 4 hours after transfection, add CHO serum-free transient feed medium CA06 at a volume ratio of 5%. After culturing for 24 hours, the intermediate additive was added to supplement sugar to 12 g / L, and the culture temperature was lowered from 37°C to 33°C, and the culture was continued. After 48 hours of culture, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%, and sugar was supplemented to 12 g / L. After 72 hours of culture, sugar was added to 12 g / L. After 96 hours of culture, CHO serum-free transient feed medium CA06 was added at a volume ratio of 5%, and sugar was supplemented to 12 g / L. The sugar content was monitored daily and replenished to 12 g / L in time. The cells were harvested on the 10th day of culture.

2. A culture method suitable for high-density transient transfection of CHO cells according to claim 1, characterized in that: The CHO cells are Expi CHO-S cells.

3. The use of a culture medium suitable for high-density transient transfection of CHO cells according to any one of claims 1 to 2, characterized in that: A culture method suitable for high-density transient transfection of CHO cells as described in any one of claims 1 to 2 is used for the culture of CHO cells.

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