A culture medium for high-density serum-free suspension culture of BHK-21 cells and its application

Abstract

This invention relates to a culture medium for high-density serum-free suspension culture of BHK-21 cells and its application. The culture medium contains inorganic salts, amino acids, vitamins, energy substances, buffers, antioxidants, sterols, polyamines, additives, and indicators. The antioxidants include dihydrolipoic acid (DLA) and S-acetyl-L-glutathione. This invention provides a culture medium with clearly defined components and significant culture effects for serum-free suspension culture of BHK-21 cells. The added DLA and S-acetyl-L-glutathione, as a combination of antioxidants in the culture medium, enhance the antioxidant properties of the serum-free suspension medium while also improving the cell viability of BHK-21 cells cultured in the medium.

Description

Technical Field

[0001] This invention relates to the field of biological products, and more specifically, to a culture medium for serum-free high-density suspension culture of BHK-21 cells and its application in a porcine Seneca virus vaccine. Background Technology

[0002] BHK-21 cells are a type of baby hamster kidney cell, commonly known as Baby Hamster Kidney cell (BHK-21). These cells are fibroblasts that primarily grow adherently in vitro, but can grow in suspension after suspension acclimation. BHK-21 cells are highly sensitive to various viruses found in economically important animals and can be widely used in the preparation of vaccines against Newcastle disease virus, swine encephalitis B, foot-and-mouth disease, bovine nodular dermatosis virus, pseudorabies virus, goatpox virus, and swine Seneca virus.

[0003] The use of serum-free suspension cell culture technology offers hope for the development of high-quality vaccines. The use of serum increases the difficulty of downstream separation and purification, raising production costs. On the other hand, single-cell suspension culture is not limited by cell growth surfaces, which is more conducive to high-density cell growth and facilitates large-scale bioreactor production of cells and related products. Therefore, the development and research of a serum-free suspension culture system for BHK-21 cells, with clearly defined components and free of serum and animal-derived components, is of great significance.

[0004] Senecavirus type A (SVA) is a pathogen that causes idiopathic vesicular disease (IVD) in pigs. The clinical symptoms of SVA are similar to those of vesicular stomatitis and foot-and-mouth disease virus infection, causing vesicles to appear in the snout and mouth of pigs, which then rupture. SVA primarily infects newborn piglets, leading to acute death, with the highest mortality rate (30%–70%) in piglets aged 1–4 days. Due to the lack of effective therapeutic drugs, vaccination remains the most effective method for controlling and preventing infectious diseases. Currently, there are no highly effective commercially available vaccines, making it difficult to effectively control large-scale outbreaks. Summary of the Invention

[0005] This invention provides a culture medium for serum-free suspension culture of BHK-21 cells and its application in Seneca virus culture.

[0006] According to a first aspect of the present invention, a culture medium for serum-free suspension culture of BHK-21 cells with clearly defined components and significant culture effects is provided. Dihydrolipoic acid and S-acetyl-L-glutathione are added in a certain proportion as an antioxidant combination of the culture medium, which enhances the antioxidant properties of the serum-free suspension culture medium while also improving the cell survival rate of BHK-21 cells cultured in the medium.

[0007] Specifically, the culture medium for serum-free suspension culture of BHK-21 cells in this invention contains:

[0008] Inorganic salts: The inorganic salts include compounds containing iron, magnesium, potassium, sodium, zinc, and calcium;

[0009] Amino acids: The amino acids include L-glutamine, L-arginine hydrochloride, L-cysteine ​​hydrochloride, L-glutamate hydrochloride, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine;

[0010] Vitamins: The vitamins mentioned include nicotinamide, D-calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and folic acid;

[0011] Energy substances: The energy substances include D-glucose, sodium pyruvate, inositol, and L-glutamine;

[0012] Buffering substances: The buffering substances include sodium bicarbonate, HEPES, or MOPS;

[0013] Antioxidants: The antioxidants include dihydrolipoic acid and S-acetyl-L-glutathione;

[0014] Sterols: The sterols include cholesterol;

[0015] Polyamines: The polyamines include putrescine or spermine and spermidine;

[0016] Additives: The additives include polyvinyl alcohol, F68, dextran sulfate, choline chloride, ethanolamine, and thymidine;

[0017] Indicator: The indicator includes sodium phenol red.

[0018] This invention relates to a culture medium for serum-free suspension culture of BHK-21 cells, wherein the final concentrations of each component of the culture medium, expressed in mg / L, are as follows:

[0019] Ferric nitrate nonahydrate 10-80, dihydrolipoic acid 0.01-0.1, magnesium sulfate 10-50, choline chloride 10-100, potassium chloride 100-500, sodium selenite 10-30, sodium bicarbonate 1000-2000, ethanolamine 1-10, sodium chloride 1000-5000, ferric citrate 70-80, zinc sulfate heptahydrate 0.1-1, polyvinyl alcohol 10-30, sodium dihydrogen phosphate 10-50, F681000-2000, L-glutamine 100-500, zinc sulfate 10-50, L-arginine hydrochloride 100-500, cholesterol 2-20, L-cysteine ​​hydrochloride 10-50, S-acetyl-L-glutathione 1-5, L-glutamate hydrochloride 100-1000, dextran sulfate 10-100, glycine 10-80, folic acid 5-2 0, L-histidine hydrochloride 10-80, inositol 5-30, L-isoleucine 100-200, nicotinamide 5-20, L-leucine 100-200, D-calcium pantothenate 5-20, L-lysine hydrochloride 100-200, pyridoxal hydrochloride 5-20, L-methionine 100-200, pyridoxine hydrochloride 5-20, L-phenylalanine 100-250, riboflavin 0.2-1 L-Serine 10-100, Thiamine Hydrochloride 2-20, L-Threonine 100-250, D-Glucose 1000-5000, L-Tryptophan 10-100, Thymidine 20-80, L-Tyrosine 10-100, Sodium Phenol Red 10-30, L-Valine 10-100, Sodium Pyruvate 10-100, Putrescine 0.1-1, HEPES 1000-2000.

[0020] Preferably, the final concentration of each component of the culture medium, expressed in mg / L, is as follows:

[0021] Ferric nitrate nonahydrate 50, lipoic acid 0.05, magnesium sulfate 30, choline chloride 50, potassium chloride 300, sodium selenite 25, sodium bicarbonate 1500, ethanolamine 10, sodium chloride 4000, ferric citrate 75, zinc sulfate heptahydrate 0.5, polyvinyl alcohol 15, sodium dihydrogen phosphate 30, F68 1000, L-glutamine 400, zinc sulfate 10, L-arginine hydrochloride 500, cholesterol 15, L-cysteine ​​hydrochloride 40, S-acetyl-L-glutathione 4, L-glutamic acid hydrochloride 800, dextran sulfate 80, glycine 40, folic acid 10, L-histidine hydrochloride 80, inositol 15, L-isoleucine 150, nicotinamide 10, L-leucine 150, D-calcium pantothenate 10 L-Lysine hydrochloride 150, pyridoxal hydrochloride 10, L-methionine 100, pyridoxine hydrochloride 10, L-phenylalanine 100, riboflavin 0.5, L-serine 100, thiamine hydrochloride 10, L-threonine 200, D-glucose 5000, L-tryptophan 50, thymidine 60, L-tyrosine 50, sodium phenol red 20, L-valine 50, sodium pyruvate 50, putrescine 1, HEPES 1000.

[0022] According to a second aspect of the present invention, a method for acclimating a non-suspension BHK-21 cell line to a suspension BHK-21 cell line is provided, comprising:

[0023] The steps of acclimatizing and culturing the non-suspension BHK-21 cell line using the culture medium described above in this invention.

[0024] The method includes the following steps: cell resuscitation, cell passage culture, serum-depleted adherent cell culture, and serum-free suspension cell culture.

[0025] In the serum-free suspension culture step, the serum-free culture medium of the present invention is used.

[0026] BHK-21 adjusted the cell density of the last generation obtained in this step to 1×10⁶ cells using serum-free, fully suspended culture medium. 6 Cells were cultured at 37°C in a 5% CO2 shaker at 140 rpm for a density of 3 × 10⁶ cells / mL. Cells were passaged by centrifugation with medium exchange, without fractionation. During the initial suspension stage, cells tend to aggregate and die in large numbers. This method prevents the release of enzymes after cell disruption from affecting cell growth and avoids starting with too low a cell density, thus preventing growth failure. Cells were cultured until the density reached 3 × 10⁶ cells / mL. 6 At cells / mL, adjust the cell density to 5 × 10⁶ cells / mL using serum-free, fully suspended culture medium. 5Cells / mL were cultured at 37℃ in a shaker with 5% CO2 at 140 rpm. After 15-20 passages, the cells showed stable proliferation, uniform size, and no cell aggregation, with a viability of over 95%. A serum-free, fully suspended culture BHK-21 cell line was obtained and named BHK-21-S.

[0027] According to a third aspect of the present invention, the present invention provides a method for preparing Seneca virus using a suspension cell line, wherein the method comprises the following steps:

[0028] (1) Using the culture medium prepared by the present invention and the method of the present invention for acclimatizing the non-suspension BHK-21 cell line, the non-suspension BHK-21 cell line is acclimatized into a suspension BHK-21 cell line;

[0029] (2) Use the suspended BHK-21 cell line obtained in step (1) to prepare Seneca virus.

[0030] In some embodiments, the Seneca virus described in this invention may be selected from the group consisting of: SVV-001 strain, CH-HN strain, CH-GD strain, CH-01-2015, CH-HB-2017, CH-04-2015 strain, SCH-DB-11-2015 strain, CH-DL-01-2016 strain, CH-ZW-01-2016 strain, HB-CH-2016 strain, THA / 2016 strain, VNM / 2019 strain, KS15-01 strain, and BRA / RS / 2016 strain.

[0031] Preferably, the virus is Newcastle disease virus, swine encephalitis virus, foot-and-mouth disease virus, bovine nodular dermatitis virus, pseudorabies virus, goatpox virus, or swine Seneca virus.

[0032] In some embodiments, the preparation of Seneca virus in step (2) includes the following steps: BHK-21 cells are prepared at 0.5-1 × 10⁻⁶ cells per cell line. 6 After seeding at a density of 5–6 × 10⁶ cells / mL, the cell density grew to 5–6 × 10⁶ cells / mL after 48 hours. 6 After adjusting the cell density to 1–2 × 10⁶ cells / mL, fresh culture medium was added. 6 Adjust the pH of the cell solution to cells / mL, inoculate with Seneca virus, and harvest the virus when the cell viability is below 40%.

[0033] In some embodiments, the culture temperature of the suspended BHK-21-S cell line in step (2) is 33–37°C; in some embodiments, the culture rotation speed is 80–120 rpm.

[0034] In some implementations, in step (2), the pH of the cell fluid to be inoculated is adjusted to 7.0 to 7.6;

[0035] In some embodiments, the initial cell density in step (2) is 1–2 × 10⁻⁶ cells / year. 6 cells / mL;

[0036] Then, inoculate with Seneca virus at a volume ratio of 0.1–1%.

[0037] In some implementations, the pH of the cell sap to be inoculated can be adjusted using various alkaline reagents, such as NaHCO3 solution, NaOH solution, KOH solution, and Na2CO3 solution.

[0038] In some embodiments, the pH of the cell sap to be inoculated with the virus is adjusted using a NaHCO3 solution. In some embodiments, the concentration of the NaHCO3 solution is 2% to 8% (w / v).

[0039] The method of this invention has achieved a technological breakthrough in the preparation of Seneca virus through suspension culture and industrial production of Seneca vaccine. The method of culturing Seneca virus effectively improves the yield, viral titer, and quality stability and uniformity of the obtained virus, reduces batch-to-batch differences, and lays the foundation for accelerating the large-scale production of suspension-based Seneca vaccine.

[0040] The serum-free, fully suspended culture BHK-21 cell line provided by this invention can be used for the following applications:

[0041] 1. Virus isolation and preparation

[0042] 2. Application in non-diagnostic and non-therapeutic viral detection

[0043] 3. Preparation of products for virus detection

[0044] 4. Vaccine product preparation

[0045] 5. Application in drug screening, wherein the drug is a drug used to prevent or treat diseases caused by viral infections.

[0046] This invention develops a serum-free culture medium with clearly defined components specifically for BHK-21 suspension cells. A suspension BHK-21-S cell line was obtained through domestication from a non-suspension BHK-21 cell line, achieving a cell density of up to 1.2 × 10⁻⁶ cells / year. 7 The cell count / mL was maintained at over 95%.

[0047] BHK21 cells obtained using the culture medium of this invention exhibit increased infectivity for Seneca virus and a significantly enhanced effective antigen content of the virus, thereby achieving the goal of high-density suspension culture of BHK-21 cells and improving the production efficiency of Seneca virus. Attached Figure Description

[0048] Figure 1 Microscopic image of adherent cells in BHK-21;

[0049] Figure 2 Microscopic image of suspended cells on BHK-21;

[0050] Figure 3 BHK-21 suspension cell growth curve;

[0051] Figure 4 Cytopathic effect of BHK-21 suspension cells after inoculation with porcine Seneca virus. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0053] Example 1: Serum-free suspension culture medium for BHK-21 cells

[0054] The serum-free suspension culture medium for BHK-21 cells of the present invention is prepared by the following method.

[0055] The components of the serum-free cell culture medium were weighed, premixed, pulverized, final mixed, and dispensed according to the formula to obtain dry powder. The dry powder was dissolved in purified water at 20-25℃, the pH was adjusted to 7.2, and the volume was adjusted to obtain BHK-21 cell serum-free suspension culture medium. The final concentrations of each component are as follows (see Table 1).

[0056]

[0057] Table 1

[0058] The serum-free suspension culture medium for BHK-21 cells provided by this invention, when used according to a specific cell suspension acclimatization method, can produce high-density, high-viability BHK-21 serum-free suspension cells. Furthermore, using this serum-free suspension culture medium and the acclimatized BHK-21 cells for scale-up suspension culture in a cell reactor to prepare viruses, the cell culture passaging operation is simple and easily achieves linear scale-up, laying the foundation for the large-scale production of corresponding viral vaccines.

[0059] In addition, the culture medium with the components shown in Table 1 in Example 1 can be used in a way that allows the concentrations of each component to fluctuate around the concentrations shown in Example 1. The technical effects of the present invention can also be achieved by using the concentration ranges shown in Table 2.

[0060] Table 2

[0061]

[0062] Example 2: Serum-free suspension acclimatization of adherent BHK-21 cells

[0063] I. Experimental Materials

[0064] 1. Cells: BHK-21 adherent cells, purchased from the American Type Culture Collection (ATCC).

[0065] 2. Culture media and reagents: Calcium- and magnesium-free PBS and DMEM were obtained from Sinopharm Tianxinhe (Wuxi) Biotechnology Co., Ltd., Trpzyme® recombinant trypsin digestion solution was obtained from Gbico, and newborn calf serum was obtained from Lanzhou Rongye Biotechnology Co., Ltd.

[0066] 3. Culture containers: T75 cell culture flasks, 125mL, 250mL, and 1000mL shake flasks, all purchased from Corning Life Sciences Co., Ltd.

[0067] II. Experimental Procedure

[0068] 1. Serum-lowering acclimatization of BHK-21 adherent cells

[0069] 1) Cell thawing: Take one BHK-21 adherent cell cryovial from the liquid nitrogen container, place it in 37°C warm water and gently shake to thaw quickly, aseptically transfer to a 10ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend in DMEM + 10% NBS, seed the cells into a T25 culture flask, and incubate at 37°C with 5% CO2. The adherent cell morphology is as follows. Figure 1 As shown, the cells are spindle-shaped and plump.

[0070] 2) Cell passage culture: After the cells have grown to a confluence and formed a monolayer, discard the culture medium, wash with calcium and magnesium-free PBS, add 1 ml of recombinant trypsin digestion solution, discard the digestion solution after the cells have rounded and dispersed, add DMEM + 10% NBS to stop digestion, passage the cells in a 1:5 ratio in separate flasks, and passage BHK-21 cells 3 times in the same way.

[0071] 3) Serum-deprived adherent cell culture: The BHK-21 cells obtained above were cultured sequentially in DMEM medium containing 5%, 2%, and 1% newborn calf serum, and passaged at a ratio of 1:5. The cells were cultured in a cell culture incubator at 37°C with 5% CO2. The medium containing 5% and 2% newborn calf serum was passaged 2-3 times each, and the medium containing 1% newborn calf serum was passaged 5-8 times each, so that the cells gradually adapted to low serum adherent culture.

[0072] 4) Serum-free cell suspension culture: Adjust the cell density of the last generation obtained in this step to 1×10⁶ cells using serum-free, fully suspended culture medium. 6 Cells were cultured at 37°C in a 5% CO2 shaker at 140 rpm for 3 × 10⁶ cells / mL. Cells were then centrifuged, changed medium, and passaged without further passage until a cell density of 3 × 10⁶ cells / mL was achieved. 6 At cells / mL, adjust the cell density to 5 × 10⁶ cells / mL using serum-free, fully suspended culture medium. 5 Cells / mL were cultured at 37℃ in a shaker with 5% CO2 at 140 rpm. After 15-20 passages, cell proliferation was stable, with no cell aggregation and a viability of over 95%. A serum-free, fully suspended culture BHK-21 cell line was obtained and named BHK-21-S.

[0073] 5) Cell cryopreservation and library construction: Obtain suspension cells that grow stably in serum-free suspension medium. Centrifuge the culture at 1000 rpm for 5 min to collect cells, resuspend in BHK-21 cell serum-free medium + 7% DMSO, and adjust the cell density to 3 × 10⁶ cells / mL. 7 The fractions were dispensed into cryovials and placed in an ultra-low temperature freezer at -80°C overnight before being transferred to a liquid nitrogen tank for long-term storage.

[0074] III. Experimental Results

[0075] like Figure 2 As shown, the serum-free suspension cells of BHK-21 obtained through domestication appeared as single, round, suspended cells under an inverted microscope, without clumping or adherent cells. In contrast, Figure 1 The adherent cells shown are spindle-shaped and attached to the surface.

[0076] Figure 3The growth of these serum-free suspension cells in serum-free suspension medium is shown. Culture conditions were 37°C, 5% CO2, and 130 rpm. Peak cell density was reached after 48 hours of culture, with cell proliferation increasing 12-fold in 48 hours, exceeding that of commercially available culture media.

[0077] Example 3

[0078] The only difference between Example 3 and Example 1 is that the concentration of some of the dihydrolipoic acid in the culture medium was replaced from 0.05 mg / L to 4 mg / L, and the concentration of S-acetyl-L-glutathione was replaced from 4 mg / L to 0.05 mg / L.

[0079] Example 4

[0080] The only difference between Example 4 and Example 1 is that the concentration of some of the dihydrolipoic acid in the culture medium was replaced from 0.05 mg / L to 2.025 mg / L, and the concentration of S-acetyl-L-glutathione was replaced from 4 mg / L to 2.025 mg / L.

[0081] Comparative Example

[0082] Comparative Example 1

[0083] The difference between Comparative Example 1 and Example 1 is that some of the dihydrolipoic acid in the culture medium was replaced with lipoic acid.

[0084] Comparative Example 2

[0085] The difference between Comparative Example 2 and Example 1 is that S-acetyl-L-glutathione was not added to the culture medium, and the concentration of some dihydrolipoic acid was replaced from 0.05 mg / L to 4 mg / L.

[0086] Comparative Example 3

[0087] The difference between Comparative Example 3 and Example 1 is that no dihydrolipoic acid was added to the culture medium, and the concentration of S-acetyl-L-glutathione was replaced from 4 mg / L to 2.025 mg / L.

[0088] Culture medium performance test

[0089] BHK-21 cells acclimatized in Examples 1, 3, 4 and Comparative Examples 1, 2, 3 were cultured in serum-free suspension at a concentration of 0.5 × 10⁻⁶. 6 Cells / ml were seeded into a 10L bioreactor, with reactor parameters set as follows: temperature 37℃, rotation speed 90 rpm / min, dissolved oxygen 60%, and pH 7.0. Samples were taken every 24 hours for cell counting and viability testing.

[0090] Table 3 shows the cell density and cell viability of serum-free suspension cultured BHK-21 cells cultured for 4 days in Examples 1, 3, and 4.

[0091] Table 3

[0092]

[0093] Table 4 shows the cell density and cell viability of serum-free suspension cultured BHK-21 cells cultured for 4 days in Comparative Examples 1-3.

[0094] Table 4

[0095]

[0096] refer to Figure 2 , Figure 3 As shown in Table 3, BHK-21 suspension cells acclimated to the culture medium with the concentration of components from Example 1 appeared as single, round, suspended cells under an inverted microscope, without clumping or adherent cells. Furthermore, the BHK-21 suspension cells acclimated to the culture medium with the concentration of components from Example 1 were cultured at a concentration of 0.5 × 10⁻⁶. 6 After seeding at a cell density of 6 × 10⁶ cells / ml, BHK-21 suspension cells reached a density of 6 × 10⁶ cells / ml after 48 hours of growth. 6 The cells / ml increased 12-fold, with good cell morphology and a viability of 98%, indicating that the BHK-21 cells domesticated in the serum-free suspension culture medium of the present invention grew well and had a high proliferation density, which can meet the needs of large-scale production of BHK-21 cells.

[0097] As can be seen from Examples 1, 3, 4 and Table 3, the BHK-21 suspension cells domesticated in Example 1 showed better growth in cell density and cell viability over 4 days than those in Examples 2 and 3.

[0098] Referring to Example 1, Comparative Examples 1-3, and Tables 3 and 4, it can be seen that the BHK-21 suspension cells acclimated to the culture medium with the component concentration of Example 1 showed better cell density proliferation and cell viability over 4 days than Comparative Examples 1, 2, and 3. This may be because the lipoic acid in Comparative Example 1 was not hydrogenated, which is not conducive to the synergistic antioxidant effect with S-acetyl-L-glutathione. Comparative Example 2 lacked the antioxidant capacity to scavenge reactive oxygen species outside the cell due to the lack of S-acetyl-L-glutathione, and Comparative Example 3 lacked the antioxidant capacity to scavenge reactive oxygen species inside the cell due to the lack of partially hydrogenated lipoic acid. These factors all contribute to the poor proliferation density and cell viability of the BHK-21 suspension cells acclimated to the culture medium.

[0099] This application uses serum-free, fully suspended culture medium for BHK-21 cells, which allows the cells to proliferate at high density and high viability in a suspended state, avoiding the digestion process required for adherent cells and making it easy to achieve large-scale culture of BHK-21 cells.

[0100] Example 5: Preparation of porcine Seneca virus

[0101] Two days after BHK-21 suspension cells were grown, the cell density was adjusted to 1.5-2 × 10⁻⁶ cells using serum-free suspension medium. 6 Cells / ml, inoculated with 0.1% (v / v) virus, cultured on a shaker at 120 rpm, 37℃, and 5% CO2 for 24 hours. Harvest when cell viability is below 40% and observe for cytopathic effects. Figure 4 As shown, there are many cell fragments, some of which are aggregated and vary in shape and size. The harvested virus solution was subjected to three freeze-thaw cycles at -80℃, centrifuged at 5000 rpm for 10 min, and the supernatant virus solution was collected for testing.

[0102] Using the Reed-Muench method, via TCID 50 The test showed a viral titer of 10. 8.2 TCID 50 / 0.1mL. It is evident that the serum-free, fully suspended cultured BHK-21 cell line acclimated by this invention is sensitive to porcine Seneca virus. This indicates that the BHK-21 serum-free suspension culture medium of this invention has good application prospects in the production of porcine Seneca virus vaccines.

[0103] The above are merely embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A culture medium for serum-free suspension culture of BHK-21 cells, wherein the final concentrations of each component in the culture medium, expressed in mg / L, are as follows: Ferric nitrate nonahydrate 10-80, dihydrolipoic acid 0.01-0.1, magnesium sulfate 10-50, choline chloride 10-100, potassium chloride 100-500, sodium selenite 10-30, sodium bicarbonate 1000-2000, ethanolamine 1-10, sodium chloride 1000-5000, ferric citrate 70-80, zinc sulfate heptahydrate 0.1-1, polyvinyl alcohol 10-30, sodium dihydrogen phosphate 10-50, F68 1000-2000, L-glutamine 100-500, zinc sulfate 10-50, L-arginine hydrochloride 100-500, cholesterol 2-20, L-cysteine ​​hydrochloride 10-50, S-acetyl-L-glutathione 1-5, L-glutamate hydrochloride 100-1000, dextran sulfate 10-100, glycine 10-80, folic acid 5-20, L-histidine hydrochloride 10-80, inositol 5-30, L-isoleucine 100-200, nicotinamide 5-20, L-leucine 100-200, D-calcium pantothenate 5-20, L-lysine salt Salt 100-200, Pyridoxal hydrochloride 5-20, L-methionine 100-200, Pyridoxine hydrochloride 5-20, L-phenylalanine 100-250, Riboflavin 0.2-1, L-serine 10-100, Thiamine hydrochloride 2-20, L-threonine 100-250, D-glucose 1000-5000, L-tryptophan 10-100, Thymidine 20-80, L-tyrosine 10-100, Sodium phenol red 10-30, L-valine 10-100, Sodium pyruvate 10-100, Putrescine 0.1-1, HEPES 1000-2000.

2. The culture medium according to claim 1, wherein the final concentration of each component of the culture medium, in mg / L, is: Ferric nitrate nonahydrate 50, lipoic acid 0.05, magnesium sulfate 30, choline chloride 50, potassium chloride 300, sodium selenite 25, sodium bicarbonate 1500, ethanolamine 10, sodium chloride 4000, ferric citrate 75, zinc sulfate heptahydrate 0.5, polyvinyl alcohol 15, sodium dihydrogen phosphate 30, F68 1000, L-glutamine 400, zinc sulfate 10, L-arginine hydrochloride 500, cholesterol 15, L-cysteine ​​hydrochloride 40, S-acetyl-L-glutathione 4, L-glutamic acid hydrochloride 800, dextran sulfate 80, glycine 40, folic acid 10, L-histidine hydrochloride 80, inositol 15, L-isoleucine 150, nicotinamide 10, L-leucine 150, D-calcium pantothenate 10 L-Lysine hydrochloride 150, pyridoxal hydrochloride 10, L-methionine 100, pyridoxine hydrochloride 10, L-phenylalanine 100, riboflavin 0.5, L-serine 100, thiamine hydrochloride 10, L-threonine 200, D-glucose 5000, L-tryptophan 50, thymidine 60, L-tyrosine 50, sodium phenol red 20, L-valine 50, sodium pyruvate 50, putrescine 1, HEPES 1000.

3. A method for acclimatizing a non-suspension BHK-21 cell line into a suspension BHK-21 cell line, comprising: The step of acclimatizing and culturing the non-suspension BHK-21 cell line using the culture medium according to any one of claims 1 to 2.

4. The method according to claim 3, wherein, The method includes the following steps: cell resuscitation, cell passage culture, serum-depleted adherent cell culture, and serum-free suspension cell culture. In the serum-free cell suspension culture step, the culture medium according to any one of claims 1 to 2 is used.

5. The method according to claim 4, wherein the method comprises: In the serum-free suspension culture step, the cell density obtained from the last generation was adjusted to 1×10⁻⁶. 6 Cells were cultured at 37°C in a shaker at 140 rpm for 5% CO2. Cells were passaged by centrifugation with medium exchange, without further passage, until a cell density of 3 × 10⁶ cells / mL was reached. 6 When the cell density is adjusted to 5 × 10⁶ cells / mL using the culture medium according to any one of claims 1 to 2, the cell density is determined. 5 The cells / mL were cultured at 37℃ in a shaker with 5% CO2 at 140 rpm. After 15-20 passages, the serum-free, fully suspended culture BHK-21 cell line was obtained.

6. A method for preparing Seneca virus, comprising: (1) The non-suspension BHK-21 cell line is domesticated into a suspension BHK-21 cell line using the method of any one of claims 3 to 5; (2) Use the suspended BHK-21 cell line obtained in step (1) to prepare Seneca virus.

7. The method according to claim 6, wherein, In step (2), the culture temperature of the suspension BHK-21 cell line is 33–37℃, the culture speed is 80–120 rpm, and the pH value is adjusted to 7.0–7.

6. Initial cell density after inoculation: 1–2 × 10⁻⁶ 6 Cells / mL, then inoculated with Seneca virus at a volume ratio of 0.1–1%.

8. The method according to claim 6, wherein, The preparation of Seneca virus in step (2) includes the following steps: BHK-21 cells are prepared at 0.5-1×10⁻⁶ cells per cell line. 6 After seeding at a density of 5–6 × 10⁶ cells / mL, the cell density grew to 5–6 × 10⁶ cells / mL after 48 hours. 6 After adjusting the cell density to 1–2 × 10⁶ cells / mL, fresh culture medium was added. 6 Adjust the pH of the cell solution to cells / mL, inoculate with Seneca virus, and harvest the virus when the cell viability is below 40%.

9. The method according to any one of claims 6 to 8, wherein, The Seneca virus mentioned is selected from the following group: SVV-001 strain, CH-HN strain, CH-GD strain, CH-01-2015, CH-HB-2017, CH-04-2015 strain, SCH-DB-11-2015 strain, CH-DL-01-2016 strain, CH-ZW-01-2016 strain, HB-CH-2016 strain, THA / 2016 strain, VNM / 2019 strain, KS15-01 strain, and BRA / RS / 2016 strain.

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