A CHO-K1 cell culture medium and culture method

Abstract

The application belongs to the technical field of cell biology, and discloses a CHO-K1 cell culture medium and a culture method. The cell culture medium is a DMEM / F12 base culture medium, and 0.1-0.5 mg / L biphenylalanine short peptides are added. The culture medium can significantly improve the survival rate of CHO-K1 cells, and significantly increase the expression amount of anti-rabies virus monoclonal antibody protein.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a CHO-K1 cell culture medium and culture method. Background Technology

[0002] Chinese hamster ovary cell line K1 (CHO-K1) is a subclone of the CHO cell line established by Puck TT in 1957 from ovarian biopsy tissue of adult Chinese hamsters. This cell line is an epithelial-like adherent cell type, requires proline for proliferation, has 20-26 chromosomes, and is negative for mycoplasma. It is routinely cultured in F12K or DMEM-H medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody) at 37°C and 5% carbon dioxide. Cryopreservation requires a serum solution containing 10% dimethyl sulfoxide. As an important tool cell in the biopharmaceutical field, CHO-K1 is widely used in recombinant protein expression and gene function research.

[0003] Transient transfection (TVT) typically uses mammalian cells, primarily HEK-293 cells, as hosts due to their high expression levels and rapid protein preparation. CHO cells, as genus-derived fibroblasts, are less susceptible to human viral infection compared to HEK-293 cells. Furthermore, CHO cells are more stable in scale-up production than HEK-293 cells, making them the primary source for most marketed antibody drugs. To maintain consistency between transiently expressed proteins and those expressed in stable cell lines, more and more pharmaceutical companies are choosing CHO cells as the TVT host for antibody drugs. However, CHO cells generally exhibit lower expression levels, especially in structurally complex bispecific antibody molecules, typically below 20 mg / L, significantly limiting their use in transient transfection. Additionally, existing TVT techniques are cumbersome, often requiring cell density adjustments the day before transfection.

[0004] To this end, Chinese patent application CN118562883A discloses a method for enhancing antibody expression levels in CHO-K1 cells via transient transfection. The method includes the following steps: cell extraction and transfer; transferring CHO-K1Q cell line cryopreservation tubes to a water bath using liquid nitrogen; thawing the cryopreservation tubes in a water bath at 37.0℃±0.5℃; cell resuscitation and seeding amplification; transferring the thawed cells to preheated culture medium for passage amplification; transient transfection using PEI reagent; introducing a specific plasmid into the resuscitated and amplified cells; batch culture, adjusting the culture flexibly according to different culture conditions; and analysis using a biomolecular analyzer to obtain the data. Compared to traditional transfection methods, this method can increase antibody expression levels by 47.8%.

[0005] In addition, in recent years, researchers have attempted to enhance transient expression levels by optimizing carrier elements (such as introducing potent promoters and matrix-associated region (MAR) elements), developing novel transfection reagents (such as cationic polymer nanoparticles), or adjusting culture conditions (such as adding histone deacetylase inhibitors). However, these strategies often suffer from drawbacks such as high cost, complex processes, or batch-to-batch instability. Therefore, how to increase expression levels without affecting the growth of CHO-K1 cells has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this invention is to provide a CHO-K1 cell culture medium and a culture method. To achieve the above objective, this invention adopts the following technical solution: One aspect of this invention relates to a CHO-K1 cell culture medium, wherein the cell culture medium is based on DMEM / F12 and supplemented with 0.1-0.5 mg / L of bisphenylalanine short peptide.

[0007] In a preferred embodiment of the present invention, the amount of the bisphenylalanine short peptide added is 0.15-0.25 mg / L; preferably 0.2 mg / L.

[0008] Another aspect of the present invention relates to a method for culturing CHO-K1 cells, comprising the following steps: (1) Adherent CHO-K1 cells were cultured in complete culture medium. The specific steps included: using DMEM / F12 basal medium containing 10% fetal bovine serum as the complete culture medium; removing commercially available adherent CHO-K1 cells from the liquid nitrogen tank, rethawing them in a 37°C water bath, and placing them in preheated complete culture medium after thawing, controlling the total density to 3.0 x 10⁻⁶ cells / year. 6 cell / ml; the complete culture medium was statically cultured in a cell culture incubator at 37°C and 5% carbon dioxide until the logarithmic growth phase; (2) DMEM / F12 medium supplemented with 0.2 mg / L diphenylalanine peptide was selected as the culture medium; the CHO-K1 cells adhered in step (1) were digested with 0.25% trypsin, followed by washing twice with PBS buffer, and then the medium supplemented with diphenylalanine peptide was seeded into 125 ml shake flasks, controlling the total density to 1.0 x 10⁻⁶. 6 Cells / ml were cultured in a shaker at 37°C, 5% carbon dioxide, and 120 rpm.

[0009] In a preferred embodiment of the present invention, the culture method further includes step (3): the passage interval is 1-2 days, and after 3 passages, an expression vector for anti-rabies virus monoclonal antibody is added to the culture medium in step (2) for transfection. After 2 days of culture, the adherent CHO-K1 cells are digested with 0.25% trypsin, then washed twice with PBS buffer, and then the culture medium is seeded into 125ml shake flasks, controlling the total density to 1.0 x 10⁻⁶. 6 Cells / ml were passaged in a cell shaker at 37°C, 5% carbon dioxide, and 120 rpm.

[0010] In a preferred embodiment of the present invention, the culture method further includes the recovery of anti-rabies virus monoclonal antibody protein.

[0011] Beneficial effects: The culture medium of the present invention can significantly improve the viability of CHO-K1 cells and significantly increase the expression level of anti-rabies virus monoclonal antibody protein. Attached Figure Description

[0012] Figure 1 Microscopic image of CHO-K1 cells on day 14 of passage culture in Example 1 of this invention; Figure 2 Example 2 of this invention: Microscopic image of CHO-K1 cells on day 14 of passage culture; Figure 3 Microscopic image of CHO-K1 cells on day 14 of passage culture in Comparative Example 1 of this invention. Detailed Implementation

[0013] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] Example 1: (1) Adherent CHO-K1 cells were cultured in a complete culture medium. The specific steps included: using DMEM / F12 basal medium containing 10% fetal bovine serum as the complete culture medium; taking commercially available adherent CHO-K1 cells out of the liquid nitrogen tank, rethawing them in a 37°C water bath, and placing them in a preheated complete culture medium after thawing, controlling the total density to 3.0 x 10⁻⁶. 6 cell / ml; incubate the complete culture medium statically in a cell culture incubator at 37°C and 5% CO2 until the logarithmic growth phase.

[0015] (2) The cells from step (1) were transferred to a culture medium for suspension growth. The specific steps included: using DMEM / F12 medium supplemented with 0.2 mg / L diphenylalanine peptide as the culture medium; digesting the adherent CHO-K1 cells from step (1) with 0.25% trypsin, then washing twice with PBS buffer, and then seeding the medium supplemented with diphenylalanine peptide into a 125 ml shake flask, controlling the total density to 1.0 x 10⁻⁶. 6 Cells / ml were cultured in a shaker at 37°C, 5% carbon dioxide, and 120 rpm.

[0016] (3) The passage interval is 1-2 days. After passage 3 times, the expression vector pHu1A9 of anti-rabies virus monoclonal antibody (the preparation method of expression vector pHu1A9 has been disclosed in Chinese Patent No. CN104603149B) is added to the culture medium in step (2) for transfection. After culturing for 2 days, the adherent CHO-K1 cells are digested with 0.25% trypsin, then washed twice with PBS buffer, and then the culture medium is seeded into a 125ml shake flask, the total density is controlled at 1.0X106 cells / ml, and the cells are continued to be passaged in a cell shaker under the conditions of 37℃, 5% carbon dioxide and 120rpm shaking.

[0017] (4) On day 14, cell viability and expression levels of anti-rabies virus monoclonal antibody protein were detected, and the results are shown in Table 1.

[0018] Example 2: Same as Example 1, except that the amount of bisphenylalanine short peptide added in step (2) was 0.4 mg / L. On the 14th day, the cell viability and the expression level of anti-rabies virus monoclonal antibody protein were detected respectively. The results are shown in Table 1.

[0019] Comparative Example 1: Same as Example 1, except that the diphenylalanine short peptide was not added in step (2). On day 14, cell viability and expression level of anti-rabies virus monoclonal antibody protein were detected respectively. The results are shown in Table 1.

[0020] Table 1: Cell culture and expression results of each example and comparative example

[0021] Experimental results show that the culture medium of the present invention can significantly improve the viability of CHO-K1 cells and significantly increase the expression level of anti-rabies virus monoclonal antibody protein.

[0022] The preferred embodiments of the present invention have been described in detail above. However, the present disclosure is not limited to the specific details of the above embodiments. Any equivalent substitutions or improvements made without departing from the concept of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for culturing CHO-K1 cells, characterized in that, Includes the following steps: (1) Adherent CHO-K1 cells were cultured in complete culture medium. The specific steps included: using DMEM / F12 basal medium containing 10% fetal bovine serum as the complete culture medium; removing commercially available adherent CHO-K1 cells from the liquid nitrogen tank, rethawing them in a 37°C water bath, and placing them in preheated complete culture medium after thawing, controlling the total density to 3.0 x 10⁻⁶ cells / year. 6 cell / ml; the complete culture medium was statically cultured in a cell culture incubator at 37°C and 5% carbon dioxide until the logarithmic growth phase; (2) DMEM / F12 medium supplemented with 0.2 mg / L diphenylalanine peptide was selected as the culture medium; the CHO-K1 cells adhered in step (1) were digested with 0.25% trypsin, followed by washing twice with PBS buffer, and then the medium supplemented with diphenylalanine peptide was seeded into 125 ml shake flasks, controlling the total density to 1.0 x 10⁻⁶. 6 Cells / ml were cultured in a shaker at 37°C, 5% carbon dioxide, and 120 rpm. (3) The passage interval is 1-2 days. After 3 passages, the expression vector of anti-rabies virus monoclonal antibody is added to the culture medium in step (2) for transfection. After 2 days of culture, the adherent CHO-K1 cells are digested with 0.25% trypsin, then washed twice with PBS buffer, and then the culture medium is seeded into 125ml shake flasks, controlling the total density to 1.0X10. 6 Cells / ml were passaged in a cell shaker at 37°C, 5% carbon dioxide, and 120 rpm.

2. The cultivation method according to claim 1, characterized in that, The culture method also includes the recovery of anti-rabies virus monoclonal antibody protein.

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