CN105505853A - Low-serum culture medium for high-density suspension culture of BHK-21 cells and application of low-serum culture medium in proliferation of FMDVs (foot and mouth disease viruses) - Google Patents

Abstract

The invention discloses a low-serum medium for high-density suspension culture of BHK-21 cells and its application in the proliferation and culture of foot-and-mouth disease virus. The present invention proposes a personalized BHK-21 cell low-serum medium by studying the metabolic level and mutual regulation of nutrients such as glucose in the growth process of BHK-21 cells, including amino acid parts, vitamin parts, balanced salt parts and other additive parts . Using the medium of the present invention for high-density suspension culture of BHK-21 cells can greatly improve the effective utilization of nutrients by cells and reduce the concentration of metabolic by-products, thereby improving cell culture efficiency; in addition, using the medium of the present invention to obtain The BHK-21 cells of BHK-21 cells have increased sensitivity to different serotypes of FMDV, and the content of the effective antigen 146S of the virus has been greatly increased, which can achieve the purpose of high efficiency of cell growth, controllability of cell growth and minimum concentration of metabolic by-products, so that The production efficiency of biological products is higher, the production cost is lower, and it has significant economic benefits.

Description

A low-serum medium for high-density suspension culture of BHK-21 cells and its application in the proliferation of foot-and-mouth disease virus

technical field

The invention relates to the development of a BHK-21 cell full-suspension high-density low-serum individualized culture medium and its application in the proliferation of foot-and-mouth disease virus, belonging to the field of veterinary biopharmaceuticals.

Background technique

With the rapid development of biological product technology, the current production process of foot-and-mouth disease inactivated vaccine in my country has developed from the use of traditional spinner bottle adherent cell culture to large-scale cell suspension culture in bioreactors. The use of bioreactors for large-scale cell culture can greatly increase the unit output of large-scale animal cell culture, realize high-density culture and high-density expression of cells, simplify the production process, reduce production costs, and ensure large-scale production of products quality. However, ordinary medium cannot meet the high-density culture of cells in the reactor, nor can it support the unique culture mode of the reactor. Personalized medium has become the mainstream of medium development in foreign countries, has been widely used and has produced great economic benefits. Appropriate medium formula can increase cell culture density and cell viability, and increase virus yield. Therefore, how to develop personalized culture medium according to different large-scale cell culture processes and improve the stability and yield of target products is a research hotspot and difficulty in this field.

According to the existing cell lines, cell culture methods, target products and production processes, cell metabolism and nutrient requirements, the present invention establishes a culture medium design scheme, establishes detection methods for various nutrients, analyzes cell metabolic flow, and designs a culture medium Formula, compared with the commercial culture medium, the serum content is reduced by 30%, the effective utilization of nutrients by cells is improved, the concentration of metabolic by-products is reduced, the culture density and cell viability of BHK-21 cells are improved, and the cell production capacity is increased by 28%. After FMD virus proliferation, the sensitivity of cells to different serotypes of FMDV increases, and the antigen content is stable and increased by 43%, so as to ensure the high density of cultured cells and the production of high concentrations of virus antigens in a stable cell culture process. , to produce high-quality virus vaccines, laying the foundation for the improvement of target product quality and industrialized production.

Contents of the invention

One of the objectives of the present invention is to provide a low-serum personalized culture medium suitable for high-density suspension culture of BHK-21 cells with low cost and relatively clear components.

The second object of the present invention is to provide the use of the culture medium in high-density suspension culture of BHK-21 cells.

The third object of the present invention is to provide the use of the culture medium in virus propagation.

In order to achieve the above object, the present invention adopts the following technical means:

1. The strategy method of developing culture medium is completed by the following steps:

(1) Preparation and preparation of cell basal culture medium

Weigh the MEM medium powder and dissolve it in a certain amount of distilled water (the temperature of distilled water is lower than 40°C), stir well to dissolve, adjust the pH value to 6.95-7.15 with sodium hydroxide or hydrochloric acid, and finally make the volume to obtain BHK-21 cell suspension culture Use medium.

(2), cell recovery and culture

One cell was taken from the suspension BHK-21 cell working bank, and after resuscitation, it was cultured in a T100 cell square bottle, and passed on for 2 to 3 generations. After the cells covered a single layer, they were digested with trypsin, and an appropriate amount of cell culture medium was added and transferred to In a 1000ml Erlenmeyer flask, shake suspension culture at 37°C, the shaker speed is 90r/min, the pH value is 7.0-7.2, and the pH value is adjusted with NaHCO ₃ every day. After the cell culture volume reaches the specified number, collect the cells and transfer them to a 5L bioreactor for culture. The culture setting parameters are: temperature 37.0-37.5°C, pH value 7.0-7.2, DO value 60.0%-80.0%, stirring The rotation speed is 80-120r/min, and the culture is subcultured for 50 hours.

(3), BHK-21 cells in the control of cell growth metabolism parameters in basal medium

① Batch culture the cells obtained in the above step 2 in a 5L bioreactor, and take cell samples at 0h, 24h, 48h, 72h and 96h of culture based on the evaluation index of cell density and cell viability, and store them at 4°C, Centrifuge at 1000rpm for 5min, and collect the cell supernatant;

② The cell supernatant obtained in the above ① is subjected to the detection and analysis of the main nutrients glucose and glutamine, and the concentration of glucose and glutamine is measured by NOVAPLUS100 biochemical analyzer;

③ The cell supernatant obtained in the above ① is subjected to the detection and analysis of the main nutrient amino acid, the amino acid concentration is analyzed by the pre-column derivatization method, and the ultra-high liquid chromatograph is used for analysis, and the derivatization reagent is diluted to a certain concentration of PITC and TEA solution with acetonitrile, and set aside;

④ The cell supernatant obtained in the above ① was subjected to the detection and analysis of the main metabolic by-products ammonia and lactic acid, and the NOVAPLUS100 biochemical analyzer was used for detection.

(4) Determination of medium formula

Combining the monitoring results of cell metabolic flow in the above step (3), determine the content of various nutrients that mainly affect cell growth and metabolism, especially the concentration of glucose is not lower than 16.0mmol/L, and the concentration of glutamine is not lower than 4.5mmol/L, The concentration ratio of the two is about 3.5:1; at the end of cell culture, the amino acid is not lower than 5% of the initial content; the tolerance concentration of by-products in the process of cell metabolism is determined, that is, the concentration of lactic acid is not higher than 19mmol/L, and the concentration of ammonia is not higher than Higher than 8mmol/L, thus determined the BHK-21 cell personalized low serum medium formula, and named it ZN-MEM.

(5) Verification of the actual effect of the medium formula

5.15L and 500L bioreactor continuous subculture

Use the ZN-MEM culture solution obtained in the above step 4 to carry out suspension culture of BHK-21 cells in 5L and 500L bioreactors, set parameters: temperature is 37.0-37.5°C, pH value is 7.0-7.2, DO value is 60.0 %～80.0%, the stirring speed is 60～80r/min, culture the cell samples for 0h, 24h, 48h, 72h and 96h, centrifuge at 800r/min, keep the supernatant, use the cell analyzer to analyze, investigate the BHK-21 cells in Changes in cell density and cell viability under different scale culture conditions.

Results: The cells were subcultured for 48 hours, and the cell density was above 3.8×10 ⁶ cells/ml, and the viability was higher than 94%.

5.2 Effect of virus proliferation on cells cultured in low serum

Take the cells harvested from the 500L bioreactor in the above step 5.1, and inoculate the FMD virus type A, O and Asia1 respectively.

60.0%～70.0%, the stirring speed is 40～60r/min. When the cytopathic rate reached over 90%, the virus was harvested, the time of harvesting the virus was recorded, and the TCID ₅₀ , LD ₅₀ , and effective antigen 146S content of the harvested virus liquid were measured by conventional methods.

Results: After testing, the TCID ₅₀ and LD ₅₀ of the harvested virus liquid all met the requirements of industrialized vaccine production. Compared with the conventional method, the cytopathic time was shortened by about 14%, and the effective antigen 146S content was increased by 43%.

On the basis of the above research, the present invention proposes a low-serum medium for high-density suspension culture of BHK-21 cells, named ZN-MEM, including amino acid part, balanced salt part, vitamin part, other additives, Serum and distilled water, pH value 7.0～7.2, wherein, the added concentration of serum is 1% (v/v), the final concentration of other raw materials in the culture medium is:

①Amino acid part, including:

②Balanced salt part, including:

③Vitamin part, including:

④Other additives, including:

In the present invention, preferably, the final concentration of other raw materials in the culture medium is: ① amino acid part, including:

②Balanced salt part, including:

③Vitamin part, including:

④Other additives, including:

Furthermore, the present invention also proposes the use of the medium in the suspension culture of BHK-21 cells.

In the present invention, preferably, the culture conditions of the suspension culture are as follows: the inoculation density is 0.50× ¹⁰⁶ /ml, the culture temperature is 37.0-37.5°C, the pH value is 7.0-7.2, and the DO value is 60.0%. ～80.0%, the stirring speed is 60～80r/min, cultured for 48h for passage.

Furthermore, the present invention also proposes the use of the culture medium in virus propagation using BHK-21 cells as host cells.

In the present invention, preferably, the virus propagation includes inoculating the virus into the BHK-21 cells cultured in the medium of the present invention, and the control parameters are set as follows: the temperature is 36.5-37.0°C, and the pH value is 7.4 ~7.6, the DO value is 60.0%~70.0%, the stirring speed is 40~60r/min, and the virus multiplication culture is carried out.

Wherein, the virus is preferably foot-and-mouth disease virus, more preferably FMD/O/MYA98/BY/2010, FMD/Re-AWH/09 or FMD/Asia1JSL/06.

The present invention changes or adjusts the types, quantities and proportions of various components in the culture medium by studying the metabolism level of BHK-21 cells to nutrients such as glucose, glutamine and amino acids and the mutual regulation of multiple factors, and optimizes the cell culture environment. A personalized low-serum medium suitable for BHK-21 suspension cells is produced to ensure the production of high-quality virus vaccines under a stable cell culture process. Compared with current commercial media, its advantages are as follows:

1. Cell quality improvement

1) Increased cell production capacity

The application of the personalized medium ZN-MEM of the present invention greatly improves the effective utilization of nutrients by cells, reduces the concentration of metabolic by-products, optimizes the cell growth environment, increases the cell growth rate, and greatly increases the final density of cultured cells. From the original 2.5×10 ⁶ cells/ml to 3.8-5.0×10 ⁶ cells/ml, the cell density increased by nearly 1.5-2 times compared with the commercial medium, the cell growth state was stable, and the cell production capacity increased by more than 28%;

2) Increased cell activity

It avoids the excess or lack of nutrients, reduces the concentration of toxic metabolic by-products in the culture medium, optimizes the nutritional environment of cells, recycles effective resources, and reduces the apoptosis rate and cell viability in the process of large-scale cell culture. improve;

3) Increased cell expansion ratio

The cell growth rate is fast. The cell subculture ratio of the commercially available BH21 cell culture medium is about 1:4, while the cell proliferation ratio of the optimized medium is 1:6-1:10, which is 40% higher, and the cell yield per unit time is increased. . At the same time, the increase of the cell expansion ratio reduces the power equipment and pressurization equipment, greatly reduces the cost, and produces indirect economic benefits.

2. Improving the quality of vaccine antigens

Using BHK-21 cells as a matrix, applying the personalized culture medium of the present invention, optimizes the cell culture environment, improves the cultured cell density and cell viability, increases the cell production capacity, and the sensitivity and expression level of cells to different types of FMD virus infection The improvement makes the content of 146S of the complete virion of FMD greatly increased, and the 146S content reaches more than 2 times that of commercial culture, which lays the foundation for the improvement of the quality of FMD products.

3. The economic cost of culture medium is reduced

At present, cell production accounts for more than 80% of the direct cost of the FMD oil adjuvant inactivated vaccine used in animal FMD vaccination. In the production process of cells using conventional culture medium, the average direct cost per liter of nutrient solution is 5.8 yuan. Using the personalized medium of the present invention to cultivate cells, the cost per liter of nutrient solution is reduced to 4.9 yuan, and the cost of cells per liter of nutrient solution is reduced by about 18 yuan. %. Based on the annual production of 1.5 billion milliliters of foot-and-mouth disease vaccine, about 750 million milliliters of cell nutrient solution is needed, and the annual direct economic output is more than 600,000 yuan.

In addition, the serum concentration is 30% lower than that of the commercial medium, which indirectly reduces the production cost, reduces the pressure on the subsequent concentration and purification process, and reduces the potential biosafety risks that may exist in the vaccine production process.

4. The process of medium preparation is simplified

The preparation process of the personalized culture medium of the present invention is simple, the solubility of reagents is enhanced, and a large amount of time and labor are not required to be invested, which saves personnel input to a certain extent.

In summary, using the personalized culture medium of the present invention to cultivate cells, the cell culture characteristics are improved, the cell yield is increased, and the effective antigen content and immune effect of the vaccine are improved; the cells produced by the personalized culture medium of the present invention are used to produce foot-and-mouth disease virus, vaccine The direct cost of production is greatly reduced. At present, this medium has been successfully applied in the pilot production line of our company, and has produced significant economic benefits, and has a wide application prospect.

Description of drawings

Fig. 1 is the required amino acid species and content distribution diagram in the process of BHK-21 cell proliferation;

Figure 2 is the amount of amino acids required for cell proliferation;

Fig. 3 is the amount of glucose required for cell proliferation;

Fig. 4 is the change of concentration of by-products of cell metabolism;

Figure 5 is a graph showing the stability and passage of cultured cells under different scales;

Figure 6 is the cell morphology of BHK-21 cells in four culture media;

Fig. 7 is the growth curve of BHK-21 cell batch culture in four kinds of culture medium;

Fig. 8 is the variation of glucose and lactic acid concentration in BHK-21 batch culture process;

Fig. 9 shows the changes of glutamine and ammonia concentrations during batch culture of BHK-21.

detailed description

The present invention will be further described below in combination with specific embodiments and accompanying drawings, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 BHK-21 Cell Nutritional Metabolism Research and Determination of Medium Formula

1 material

1.1 BHK-21 cells were taken from the BHK-21 suspension cell working cell bank established by Zhongnong Weite Biotechnology Co., Ltd., the batch number is BHK-21/W/S-201202, and they were used after the scale-up culture inspection passed.

1.2 Serum Newborn bovine serum was purchased from a qualified supplier and used after passing the internal control quality standard for newborn bovine serum of Zhongnong Weite Biotechnology Co., Ltd.

1.3 Reagents and equipment

Main reagents: American waters amino acid analysis column (5μm, 150×4.6mmol); 20 amino acid standard samples; acetonitrile (chromatographically pure); sodium acetate (analytical pure); acetic acid (analytical pure); triethylamine (TEA); Phenylisothiocyanate (PITC), etc.

Main instruments: cell analyzer (Innovatis, Germany); American Waters high-performance liquid chromatography; NOVAPLUS100 biochemical analyzer; 5L bioreactor (US NBS); microscope (Japan Olympus); 5L bioreactor (US NBS),

1.4 Cell basal culture medium: Weigh a certain amount of MEM medium powder and dissolve it in a certain amount of distilled water (distilled water temperature is lower than 40°C), stir well to dissolve, adjust the pH value to 6.95-7.15 with sodium hydroxide or hydrochloric acid, and finally make up to volume The basal culture medium for suspension culture of BHK-21 cells was obtained.

2 methods

2.1 Main measurement methods

2.1.1 Cell counting and analysis

2.1.1.1 Sampling: absorb the cells with a sterile pipette;

2.1.1.2 Adding samples: Gently pipette the cell suspension, use a pipette to absorb 20ul of the cell sample and mix it repeatedly with 10ml of CASYTON cell analysis buffer, and analyze it with a cell analyzer.

2.1.1.3 Detection: Calculate the cell viability, density and cell growth ratio according to the results measured by the cell analyzer to determine the proliferation ability of the cells.

2.1.2 Amino acid detection and analysis: detection by HPLC

2.2 Cell culture and methods

2.2.1 Cell Recovery and Culture

①Take one cell from the suspended BHK-21 cell working bank, and quickly place it in a 40°C water bath to thaw;

②Centrifuge the cells obtained in ① at 1000rpm for 4min, discard the supernatant, add an appropriate amount of culture medium containing 5% serum to resuspend, and culture it in a T100 cell square bottle after recovery, pass 2 to 3 generations, and the cells Digest with trypsin after overgrown monolayer;

③Transfer the cells obtained in ② into a 250ml Erlenmeyer flask, shake and suspend culture at 37°C, the shaker speed is 90r/min, the pH value is 7.0-7.2, adjust the pH value with NaHCO ₃ every day, and culture for 50h for subculture;

④ When the cell culture volume reaches 100ml, the cell density is 3.0×10 ⁶ cells/ml, and the cell viability is above 93%, scale up step by step, collect the cells, transfer them to a 5L bioreactor for cultivation, and set the parameters: the temperature is 37.0～37.5°C , the pH value is 7.0-7.2, the DO value is 60.0%-80.0%, the stirring speed is 80-120r/min, and the culture is subcultured for 50h.

2.2.2 Requirements of amino acids, glucose and metabolic by-products in BHK-21 cells

1) Carry out batch culture in 5L bioreactor according to the cells obtained in ④ in the above step 2, and take cell samples at 0h, 24h, 48h, 72h and 96h of culture with the evaluation index of cell density and cell viability, and in 4 Centrifuge at 1000rpm for 5min at ℃, and collect the cell supernatant;

2) The cell supernatant obtained in the above 1) is subjected to the detection and analysis of the main nutrients glucose and glutamine, and the concentration of glucose and glutamine is measured by NOVAPLUS100 biochemical analyzer;

3) The cell supernatant obtained in the above 1) is subjected to the detection and analysis of the main nutrient amino acid, and the concentration of the amino acid adopts the ultra-high liquid phase pre-column derivatization method, and the specific steps are:

①Sample pretreatment: centrifuge the cell culture medium to be tested at 1000r/min, 4°C for 10min, and take the supernatant for later use;

②Preparation of derivatization reagents: take 12uL of PITC and 144uL of TEA and dilute to 1000uL with acetonitrile, and set aside;

③ Sample derivatization reaction: Take 200uL of the supernatant, and at the same time take 100uL of PITC and TEA respectively, vortex for 30s, extract at room temperature for 1h, immediately stop with an equal volume of n-hexane for 10min, take the supernatant and filter it with a 0.22μm filter membrane, and set aside. The derivation method of the sample is the same as that of the standard;

④Mobile phase: two solutions of A and B for gradient elution. Solution A is sodium acetate solution; solution B is acetonitrile solution, and solution C is washing solution, that is, 20% acetonitrile solution; the mobile phase is filtered through a 0.22 μm filter membrane and degassed by ultrasonic waves for 10 minutes before use;

5. standard curve is formulated: the standard solution containing 20 kinds of amino acids is diluted, and concentration is respectively 0.1mmol/L, 0.25mmol/L, 0.5mmol/L, 1.0mmol/L;

6. Equilibrate the chromatographic column: first use liquid C to clean the amino acid analysis column, then use liquid A to balance the chromatographic column until the baseline is balanced;

⑦ Run the sample: the loading volume is 20uL, the flow rate is 1ml/min, the running time is 65min, the column temperature is 30°C, and the detection wavelength is 254nm;

⑧Rinse the instrument and chromatographic column, the washing sequence is more than 60ml of liquid C, and more than 30ml of liquid B;

⑨Data processing, calculation of amino acid content.

2.2.3 Optimization and application of medium formula

According to the cell growth and metabolism parameters detected in the above step 2.2.2, combined with the monitoring results of the cell metabolic flow, determine the content of each nutrient that mainly affects the cell growth and metabolism, so that the content of the nutrient is not less than 5% at the end of the cell culture , the by-product ammonia concentration in the process of cell metabolism is less than 8mM, and the lactic acid concentration is less than 19mM tolerable concentration limit, and the main nutrients are supplemented or reduced, thereby adjusting the basic catalog medium MEM.

3 results

3.1 Effects of amino acids, glucose and metabolic by-products on cell proliferation Measure the contents of 20 amino acids in the culture medium at 0h and 48h after cell inoculation by HPLC, and combine the cell growth and metabolism levels to determine the amount of various amino acids required for each cell growth , as shown in Figure 1, and then monitor the metabolism of amino acids in the process of cell growth.

Changes in the amount of amino acids, glucose, and by-products of cell metabolism required for cell proliferation are shown in Figure 2-Figure 4, respectively.

3.2 Changes in the concentrations of major metabolites during batch culture of BHK-21 cells

Table 1 Changes in concentration of main metabolites during batch culture

3.3 Confirmation and verification of medium formula

Combined with the monitoring results of the above-mentioned cell metabolic flow, determine the content of various nutrients that mainly affect cell growth and metabolism and the tolerance concentration of by-products in the process of cell metabolism to design a medium formula, named ZN-MEM, the medium includes Amino acid part, balanced salt part, vitamin part, other additives, serum and distilled water, pH value 7.0-7.2, wherein, the concentration of serum added is 1% (v/v), and the final concentration of other raw materials in the medium is as follows Table 2 shows:

Table 2

Application of embodiment two personalized culture medium under different culture scales

1 material

1.1 BHK-21 cells were taken from the working cell bank of BHK-21 suspension cells established by Zhongnong Weite Biotechnology Co., Ltd., the batch number is BHK-21/W/S-201201, and they were used after they passed the scale-up culture test.

1.2 Serum Newborn bovine serum was purchased from a qualified supplier and used after passing the internal control quality standard for newborn bovine serum of Zhongnong Weite Biotechnology Co., Ltd.

1.3 Medium

In the BHK-21 low serum medium ZN-MEM of the present invention, the medium includes amino acid part, balanced salt part, vitamin part, other additives, serum and distilled water, with a pH value of 7.0 to 7.2, wherein the added concentration of serum is 1 %(v/v), the final concentration of other raw materials in the medium is shown in Table 3 below:

The developed ZN-MEM medium formula is entrusted to the medium manufacturer for processing. Taking the preparation of 50L culture medium as an example, the preparation method is as follows:

①Measure 90%-95% of the total volume of water for injection, and when the temperature drops to 35-40°C, weigh an equivalent of 50L dry powder medium, that is, 0.925kg dry powder (does not contain glutamine, PluronicF-68 and serum) dissolved in Wherein, stirring for at least 50 minutes to fully dissolve it;

② Add 36.5 g of glutamine to the solution obtained in the above step ①, and stir for 15 minutes;

③ Add 50.0 g of anti-shear protection agent PluronicF-6850.0 g to the solution obtained in the above step ②, and stir for 15 minutes;

④ Add 1% (v/v) serum to the solution obtained in the above step ③, and mix well;

⑤ Add a certain amount of 4.0mol/L sodium hydroxide or 1.0mol/L hydrochloric acid solution to the solution obtained in the above step ④, and adjust the pH value to 7.0-7.2;

⑥ Add cooled water for injection to the solution obtained in the above step ⑤, and set the volume to a final volume of 50L;

⑦ The solution obtained in the above step ⑥ is sterilized and filtered with a filter element with a pore size of 0.22 μm, and stored at 4°C.

1.4 Reagents and equipment

Main reagents: American Waters amino acid analysis column (5μm, 150×4.6mmol); 20 amino acid standard samples; acetonitrile (chromatographically pure); sodium acetate (analytical pure); acetic acid (analytical pure); triethylamine (TEA); Phenylisothiocyanate (PITC);

Main instruments: cell analyzer (Innovatis, Germany); microscope (Olympus, Japan); American Waters high-performance liquid chromatography; NOVAPLUS100 biochemical analyzer, 5L bioreactor (US NBS), 500L bioreactor (independently designed and commissioned bioreactor device manufacturers).

2 methods

2.1 Cell count and analysis

2.1.1 Sampling: absorb the cells with a sterile pipette;

2.1.2 Adding samples: pipette the cell suspension gently, use a pipette to mix 20ul of cell samples and 10ml of CASYTON cell analysis buffer repeatedly, and analyze with a cell analyzer.

2.1.3 Detection: Calculate the cell viability, density and cell growth ratio according to the results measured by the cell analyzer to determine the proliferation ability of the cells.

2.2 Cell Recovery and Culture

①Take one cell from the suspended BHK-21 cell working bank, and quickly place it in a 40°C water bath to thaw;

②Centrifuge the cells obtained in ① at 1000rpm for 4min, discard the supernatant, add an appropriate amount of culture medium containing 5% serum to resuspend, and culture it in a T100 cell square bottle after recovery, pass 2 to 3 generations, and the cells Digest with trypsin after overgrown monolayer;

③Transfer the cells obtained in ② into a 250ml Erlenmeyer flask, culture in suspension at 37°C with shaking, the speed of the shaker is 90r/min, the pH value is 7.0-7.2, adjust the pH value with 8% NaHCO ₃ every day, until the cell culture volume After the specified number is reached, the cells are collected for subsequent cell seeding.

2.3 Subculture of BHK-21 cells in 5L bioreactor

Transfer the cells collected in the above 2.2 into a 5L bioreactor for culture, the inoculation density is 0.50×10 ⁶ cells/ml, the culture volume is 4000ml, and the culture setting parameters are: temperature 37°C, DO value 68%, pH value 7.0, The speed is 120r/min, all controlled automatically. Cultured for 48 hours, subcultured for 40 days (20 generations).

2.4 Subculture of BHK-21 cells in a 500L bioreactor

The cell seeds collected above were expanded and cultured on a 500L bioreactor, the inoculation density was 0.55×10 ⁶ cells/ml, and the reactor control parameters were: temperature 37.0°C, DO value 70.0%, pH value 7.1, speed 100r/min , are automatically controlled. Cultured for 48 hours, subcultured for 40 days (20 generations).

3. Results

Combined with the monitoring results of cell metabolic flow, the content of various nutrients that mainly affect cell growth and metabolism and the tolerance concentration of by-products in the process of cell metabolism are determined. The medium is used to verify the application of 5L and 500L cell suspension culture in different scales. The stability and passage of cells cultured on a large scale is shown in Figure 5, which shows that BHK-21 cells were continuously cultured for 40 days (20 generations) using the medium determined by the present invention, and the density was all above 3.5×10 ⁶ cells/ml, and their viability The rates are all higher than 94%. Therefore, this medium ZN-MEM can be applied to large-scale cultivation of BHK-21 cells.

Effect of BHK21 cells cultured in different mediums on virus proliferation in embodiment three

1 Materials and methods

1.1 Materials

1.1.1 Cells

BHK-21-P cells were purchased from ATCC, source: HamsterSyrianKidney, preserved by Zhongnong Weite Biotechnology Co., Ltd., and the test cells have been passed to F18 generation.

1.1.2 Medium

The commercialized low-serum medium of BHK-21 cells, namely: BHK-21MEM, BHK-21GMEM, SLMBHK-21, are named as A, B and C medium respectively; the medium ZN-MEM of the present invention is named as D medium , the formulation is identical to the medium in Example two.

1.1.3 Medium supplements

Glutamine, Pluronic F-68, ferric citrate, transferrin, putrescine, hormones, and lipids were purchased from SIGMA Company, and soybean protein hydrolyzate, ultrafiltration plant peptone, yeast extract, etc. were purchased from Beijing Reagent Company.

1.1.4 Other reagents and equipment

Commonly used reagents such as sodium bicarbonate, sodium hydroxide, and hydrochloric acid are domestic analytically pure,

Main instruments: cell counter (Innovatis, Germany), NOVAPLUS100 biochemical analyzer (NovaBiomedical, USA), inverted microscope (Olympus, Japan), etc.

1.2 Method

1.2.1 Recovery and suspension culture of BHK-21 cells

①Take one cell from the suspended BHK-21 cell working bank, and quickly place it in a 40°C water bath to thaw;

②Centrifuge the cells obtained in ① at 1000rpm for 4min, discard the supernatant, add an appropriate amount of culture medium containing 5% serum to resuspend, and culture it in a T100 cell square bottle after recovery, pass 2 to 3 generations, and the cells Digest with trypsin after overgrown monolayer;

③Transfer the cells obtained in ② into a 250ml Erlenmeyer flask, culture in suspension at 37°C with shaking, the shaker speed is 90r/min, the pH value is 7.0-7.2, adjust the pH value twice a day with 8% NaHCO ₃ , and wait for the cell culture When the volume reaches 100ml, take the above-mentioned cells in the logarithmic growth phase and transfer them to a 1000ml Erlenmeyer flask at a ratio of 1:6 to 1:10 for expansion.

1.2.2 Batch culture of BHK-21 cells in different media

Take well-grown serum-free BHK-21 suspension cells and inoculate them in four mediums A, B, C and D at the same initial density of 0.60×10 ⁶ cells/ml, and inoculate them at 37°C, 100r/min, and pH Batch culture was carried out under 7.0 culture conditions. Cell samples were taken after 0h, 24h, 48h, 72h, 96h and 120h of culture, observed under an inverted microscope, and cell growth was analyzed using cell density and cell viability as evaluation indicators.

1.2.3 Determination of cell metabolism parameters such as main nutrients and metabolic by-products

Take the cell samples of 0h, 24h, 48h and 96h in the above batch culture process, centrifuge at 800r/min, keep the supernatant, and use NOVAPLUS100 biochemical analyzer to analyze the BHK-21 cells under different serum-free medium conditions. Glutamine, glucose, ammonia, and lactic acid concentrations in the culture medium.

1.2.4 Effects of BHK21 cells cultured in different media on the proliferation of foot-and-mouth disease virus

Take the cells cultured in medium A, B, C and D, and inoculate the Re-AWH/09 type, O/MYA98/BY/2010 and FMD/Asia1JSL/06 strains of foot-and-mouth disease virus respectively, and set the control parameters: the temperature is 36.5~ 37.0°C, pH value 7.4-7.6, DO value 60.0%-70.0%, stirring speed 40-60r/min. When the cytopathic rate was above 90%, the virus was harvested, the time of harvesting the virus was recorded, and the TCID ₅₀ , LD ₅₀ and effective antigen 146S content of the harvested virus liquid were measured by conventional methods.

2 results

2.1 Comparison of cell culture morphology of BHK-21 cells in different serum-free media

The cell morphology of BHK-21 cells in the four culture media is shown in Figure 6. It can be seen that BHK-21 cells are cultured in the above four culture media, and the cell morphology is quite different. Among them, the cell morphology of the optimized culture medium in Figure D Better than other mediums A, B and C, the cells are bright, plump, round, with clear borders, and the cell size is normal (generally 10.0-11.0 μm).

2.2 Comparison of growth curves of BHK-21 cells in batch culture in different media

The growth curves of BHK-21 cells cultured in batch in four media are shown in Fig. 7 . It can be seen from Figure 7 that during the whole process of batch culture, the optimized D medium had the fastest cell growth and high cell viability. After culturing for 72 hours, the maximum viable cell density reached 8.42×10 ⁶ cells/ml, and the cell viability 96.88%. It can be seen that the optimized D medium is more suitable for BHK-21 cell culture.

2.3 Determination of cell metabolism parameters such as main nutrients and metabolic by-products

NOVAPLUS100 biochemical analyzer was used to measure the concentration of glutamine, glucose, ammonia and lactic acid in the supernatant of cells cultured for 0h, 24h, 48h and 96h.

Figure 8 shows the changes of glucose and lactic acid concentrations during BHK-21 batch culture, and Figure 9 shows the changes of glutamine and ammonia concentrations during BHK-21 batch culture.

It can be seen from Figures 8 and 9 that under the culture conditions of four mediums for BHK-21 cells, the concentration of the main nutrients glucose and glutamine in the cell culture medium gradually decreased with the prolongation of the culture time, showing a decrease in the early stage of culture compared with that in the culture period. The trend of slowing down in the fast, middle and late stages, the concentration of lactic acid and ammonia in the cell culture supernatant both accumulated continuously with the prolongation of the culture time.

By comparison, it was found that the concentration of glucose and glutamine in D medium was low, and the concentration of its metabolic by-products ammonia and lactic acid was also low (when the concentration of lactic acid and ammonia reached 19mM and 8mM, the growth of BHK-21 cells was severely inhibited effect), indicating that the concentration ratio of glucose and glutamine in D medium is optimized, thereby optimizing the cell metabolic pathway and promoting cell proliferation.

2.4 Effects of BHK-21 cells cultured in different media on the proliferation of foot-and-mouth disease virus

The BHK-21 cell that table 3 different culture medium is cultured is to the impact of foot-and-mouth disease virus proliferation

The results in Table 3 show that: the TCID ₅₀ and LD ₅₀ of the BHK-21 suspension cells cultured with the above four mediums to produce the FMD virus solution all meet the requirements of industrial production, while the cells of different types of FMD virus produced by the self-made low serum medium D The lesion time is shortened, and the effective antigen 146S content is increased by 43%, which is better than A, B, and C commercial media, which lays the foundation for the production of high-quality FMD vaccines.

3 Conclusion

BHK-21 cells were cultured with self-developed personalized medium and three commercial low-serum mediums, and the growth and virus proliferation of BHK-21 cells in different mediums were studied. The results showed that BHK-21 cells were in Cultured in the optimized D medium, which is better than A, B, and C commercial medium, the cultured cells are plump and bright, the cell growth rate is fast, the concentration of ammonia and lactic acid by-products of cell metabolism is low, and it can be continuously and stably subcultured. The density is increased by about 28%, and the effective antigen content of the produced foot-and-mouth disease virus liquid is increased by 43%, which can ensure the production of high-quality vaccines.

Claims (8)

1. the low blood serum medium for BHK-21 high cell densities suspension culture, it is characterized in that comprising amino acid moiety, balanced salt part, vitamin moieties, other additives, serum and distilled water, pH value 7.0 ~ 7.2, wherein, it is 1% (v/v) that serum adds concentration, and other each raw materials final concentration is in the medium:

1. amino acid moiety, comprising:

2. balanced salt part, comprising:

3. vitamin moieties, comprising:

4. other additive, comprising:

2. a kind of low blood serum medium for BHK-21 high cell densities suspension culture according to claim 1, it is characterized in that comprising amino acid moiety, balanced salt part, vitamin moieties, other additives, serum and distilled water, pH value 7.0 ~ 7.2, wherein, it is 1% (v/v) that serum adds concentration, and other each raw materials final concentration is in the medium:

1. amino acid moiety, comprising:

2. balanced salt part, comprising:

3. vitamin moieties, comprising:

4. other additive, comprising:

3. the purposes of the substratum described in claim 1 or 2 in the suspension culture of carrying out BHK-21 cell.

4. purposes according to claim 3, is characterized in that described suspension culture, and its culture condition is: inoculum density is 0.50 × 10 ⁶individual/ml, culture temperature is 37.0 ~ 37.5 DEG C, and pH value is 7.0 ~ 7.2, DO value is 60.0% ~ 80.0%, and mixing speed is 60 ~ 80r/min, cultivates 48h and goes down to posterity.

5. the substratum described in claim 1 or 2 is carrying out the purposes in virus multiplication using BHK-21 cell as host cell.

6. purposes according to claim 5, it is characterized in that described virus multiplication comprises the BHK-21 cell virus inoculation to obtaining through the culture medium culturing described in claim 1 or 2, setup control parameter is: temperature 36.5 ~ 37.0 DEG C, pH value is 7.4 ~ 7.6, DO value is 60.0% ~ 70.0%, mixing speed is 40 ~ 60r/min, carries out virus multiplication cultivation.

7. purposes according to claim 6, is characterized in that described virus is foot and mouth disease virus.

8. purposes according to claim 7, is characterized in that described foot and mouth disease virus is FMD/O/MYA98/BY/2010, FMD/Re-AWH/09 and FMD/Asia1JSL/06 strain.