Vero cell serum-free medium and application thereof

Abstract

The invention relates to a Vero cell serum-free culture medium and application thereof, the serum-free culture medium is suitable for free suspension growth of Vero cells and is free of animal sources and protein components, adherent cells can be directly domesticated through a certain method to screen out Vero suspension cells, and serum-free suspension culture of the Vero suspension cells is achieved in the serum-free culture medium. The density of cells subjected to suspension culture by adopting the serum-free culture medium can reach more than 107 cells / mL, the cell viability is more than 90%, the cells are well dispersed, the cells can still normally grow and proliferate after continuous subculture for 20 generations, and the cells are sensitive to various viruses.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell culture, and particularly relates to a serum-free culture medium for Vero cells and application thereof. BACKGROUND

[0002] Vero cells are African green monkey kidney cells, which can be cultured in vitro and grow adherently and can be subcultured continuously. Vero cells are sensitive to a variety of human and animal viruses, such as Rabies Virus, Poliovirus, Coronavirus, Rotavirus, Influenza Virus, Porcine epidemic diarrhea virus (PEDV), Pseudorabies Virus (PRV), etc., and are widely used in virus isolation and culture and vaccine production.

[0003] Since Vero cells have strong adherence dependence (i.e., in general, in vitro culture can only adhere to the surface of a fixed medium to grow and proliferate), in vitro culture depends on culture media (such as square bottles, cell factories, spinner flasks, microcarriers, sheet carriers, etc.).

[0004] The traditional virus culture preparation process is generally as follows: cells are cultured using a traditional culture medium (such as MEM, DMEM, M199) with the addition of 5-10% serum, the culture solution is discarded after the cells are fully grown, a cell digestion solution (such as trypsin) is added to make the cells fall off and disperse for subculture or expansion, the culture solution is discarded after the expansion is scaled up to a certain scale, and the relevant virus is inoculated to carry out virus culture, and then the corresponding virus is harvested. However, in the process of adherent culture in the conventional culture medium with the addition of a certain concentration of serum, on the one hand, serum is a mixture with unclear components, which is prone to contamination risks of exogenous viruses, mycoplasma, etc., and the batch difference of serum is large, which is easy to cause cell growth fluctuation and unstable virus yield; on the other hand, the adherent cell culture process is complex, and the cell digestion operation is required to scale up the culture scale, and the scale-up is limited by the surface area of the culture medium.

[0005] Large-scale suspension culture technology of animal cells has been applied in the production of some vaccines and antibodies, such as BHK21 cell suspension culture for producing foot-and-mouth disease virus and rabies virus, MDCK cell serum-free suspension culture for producing influenza virus and avian influenza virus, and the original cells of BHK21 and MDCK are adherent-dependent cells, which can obtain corresponding suspension cells under suitable culture medium and cell adaptation screening conditions, and then realize suspension culture medium or even serum-free suspension culture.

[0006] Serum-free suspension culture has the advantages of simple process operation, easy scale-up and lower cost. However, the existing serum-free medium still has significant deficiencies. On the one hand, most serum-free media only support adherent culture and cannot realize true free growth in suspension, on the other hand, the medium components are complex and the effect is limited, and the cell density is usually ≤5×10 6 / mL. SUMMARY

[0007] Therefore, the present application provides a Vero cell serum-free medium and its application. The Vero cell can realize free growth in suspension, thereby solving or at least alleviating one or more of the above problems and other problems in the prior art.

[0008] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is:

[0009] A Vero cell serum-free medium comprises the following components:

[0010] Component Content (mg / L) Component Content (mg / L) Calcium chloride, anhydrous L-Tyrosine Copper sulfate pentahydrate 50-200 L-Valine 20-100 Ferric nitrate nonahydrate 5-50 L-Hydroxyproline 50-200 Ferrous sulfate heptahydrate 10-50 D-Glucose 20-100 Potassium chloride 0.1-0.5 HEPES 2000-8000 Sodium metasilicate nonahydrate 200-500 Linoleic acid 2000-5000 Ammonium metavanadate 10-150 Thioctic acid 0.01-0.05 Magnesium sulfate, anhydrous 0.01-1 1,4-Butanediamine dihydrochloride 0.05-0.1 Sodium chloride 20-100 Sodium pyruvate 0.002-0.1 Sodium phosphate dibasic, anhydrous 3000-8000 Biotin 50-600 Disodium hydrogen phosphate 50-100 Calcium D-pantothenate 0.01-0.2 Zinc sulfate heptahydrate 50-100 Choline chloride 2-10 Potassium phosphate dibasic, anhydrous 0.2-2 Folic acid 10-50 Sodium selenite 50-100 i-Inositol 1-10 Ferrous ammonium citrate 20-100 Nicotinamide 1-50 L-Arginine hydrochloride 10-500 Pyridoxal hydrochloride 1-10 L-Cystine hydrochloride 150-600 Pyridoxine hydrochloride 1-10 L-Glutamine 10-80 Riboflavin 1-10 Glycine 300-1500 Thiamine hydrochloride 0.1-1 L-Histidine hydrochloride 10-100 Thymidine 1-10 L-Isoleucine 10-150 Vitamin E 0.1-1 L-Leucine 20-150 Vitamin B12 0.3-3 L-Lysine hydrochloride 50-250 Triiodothyronine (T3) 0.1-1 L-Methionine 50-150 Hydrocortisone 0.001-0.01 L-Phenylalanine 20-150 Recombinant human insulin 0.1-10 L-Serine 20-250 Ethanolamine 2-50 L-Threonine 50-150 Hypoxanthine 1-10 L-Alanine 50-200 EDTA 2-20 L-Asparagine 1-100 Glutathione 0.01-0.1 L-Aspartate 10-100 EGF 0.5-3 L-Cysteine hydrochloride 10-100 Cholesterol 0.001-0.01 L-Glutamic acid 20-150 L-Proline 1-10 Dextran sulfate 5-60 F68 500-2000 L-Tryptophan 20-200 Sodium bicarbonate 10-100 Figure 1 20-200 Figure 2 1500-3500

[0011] In the foregoing Vero cell serum-free medium, optionally, the serum-free medium comprises the following components:

[0012]

[0013]

[0014] In the foregoing Vero cell serum-free medium, optionally, the Vero cell serum-free medium comprises the following components:

[0015]

[0016]

[0017] The second technical scheme adopted by the present application is the application of the foregoing Vero cell serum-free medium in domesticating Vero adherent cells into serum-free suspension cells.

[0018] The third technical scheme adopted by the present application is a method for domesticating Vero adherent cells into serum-free suspension cells, comprising the following steps:

[0019] (1) After dispersing the monolayer Vero adherent cells, adhere to the first serum-free medium for 1-5 generations;

[0020] (2) The cells obtained after step (1) are digested and collected, and cultured in suspension for 3 to 5 generations in a culture medium containing a second serum-free medium and an anti-cell clumping agent. Then, they are cultured in suspension in the second serum-free medium to obtain domesticated Vero cells, wherein the second serum-free medium is the aforementioned serum-free medium for Vero cells.

[0021] Optionally, in step (1), the first serum-free culture medium is VP-SFM serum-free culture medium.

[0022] Optionally, in step (1), the adherent culture is carried out in a culture flask, and the inoculation density of the culture flask is (1~10)×10⁻⁶. 6 75 bottles per unit.

[0023] Optionally, in step (2), the suspension culture is carried out in a shake flask, and the inoculation density of the shake flask is (0.3~1.2)×10⁻⁶. 6 The shaker was operated at 130±5 rpm and the temperature was 37±1℃.

[0024] The fourth technical solution adopted in this invention is: the application of the aforementioned Vero cell serum-free culture medium in virus amplification.

[0025] Optionally, the virus is a sensitive virus for Vero cells, including rabies virus, poliovirus, coronavirus, rotavirus, influenza virus, porcine epidemic diarrhea virus, pseudorabies virus, and other sensitive viruses.

[0026] The fifth technical solution adopted in this invention is: a method for amplifying a virus, comprising the following steps:

[0027] The domesticated Vero cells were amplified by suspension culture using the aforementioned serum-free culture medium, and then directly inoculated with the virus for further culture.

[0028] Optionally, when the cell density in the cell culture medium reaches (1-5)×10 6 When the concentration is above 0.0001 / mL, inoculate the virus with an MOI of 0.0001-10, and control the culture temperature at 30-37.5℃, pH 6.8-7.5, DO 20-80%, and rotation speed at 30-200 rpm.

[0029] Optionally, the virus may be other susceptible viruses such as porcine pseudorabies virus, rabies virus, poliovirus, coronavirus, rotavirus, influenza virus, and porcine epidemic diarrhea virus.

[0030] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0031] The inventors of the present application develop a serum-free culture medium suitable for Vero cell suspension free growth through a large number of experimental researches, which is free of animal sources and protein components. The adherent cells can be directly domesticated to select Vero suspension cells through a certain method, so that the Vero cells can be cultured in the serum-free culture medium.

[0032] The cell density cultured in the serum-free culture medium can reach more than 10 7 cells / mL, the cell viability is more than 90%, the cells are well dispersed, and the cells can still grow and proliferate normally after continuous subculture for 20 generations. In addition, the cells are sensitive to a plurality of viruses. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 3 The inverted microscope photograph of Vero cells (P10) obtained from the domestication experiment of Example 2 at 100 times magnification.

[0034] Figure 4 The growth curve of Vero cells obtained from the domestication experiment of Example 2 using the serum-free culture medium of Example 1 is shown in the schematic diagram.

[0035] Figure 5 The growth situation of Vero cells obtained from the domestication experiment of Example 2 using the serum-free culture medium of Example 1 after continuous subculture is shown in the schematic diagram.

[0036] Component Content (mg / L) The inverted microscope photograph of Vero cells with lesions after inoculation of porcine pseudorabies virus in Example 3 is shown in the schematic diagram.

[0037] Component Content (mg / L) The culture time and virus titer of Vero cells with lesions after inoculation of porcine pseudorabies virus in Example 3 are shown in the schematic diagram. DETAILED DESCRIPTION

[0038] The present application provides a serum-free culture medium for Vero cells, which is free of animal sources and protein components. The adherent cells can be directly domesticated to select Vero suspension cells, and then the Vero cells can be cultured in the serum-free culture medium.

[0039] The serum-free culture medium for Vero cells comprises the following components:

[0040] Calcium chloride, anhydrous L-Tyrosine Copper sulfate pentahydrate L-Valine Ferric nitrate nonahydrate 50-200 L-Hydroxyproline 20-100 Ferrous sulfate heptahydrate 5-50 D-Glucose 50-200 Potassium chloride 10-50 HEPES 20-100 Sodium metasilicate nonahydrate 0.1-0.5 Linoleic acid 2000-8000 Ammonium metavanadate 200-500 Thioctic acid 2000-5000 Magnesium sulfate, anhydrous 10-150 1,4-Butanediamine dihydrochloride 0.01-0.05 Sodium chloride 0.01-1 Sodium pyruvate 0.05-0.1 Sodium phosphate dibasic, anhydrous 20-100 Biotin 0.002-0.1 Disodium hydrogen phosphate 3000-8000 Calcium D-pantothenate 50-600 Zinc sulfate heptahydrate 50-100 Choline chloride 0.01-0.2 Potassium phosphate dibasic, anhydrous 50-100 Folic acid 2-10 Sodium selenite 0.2-2 i-Inositol 10-50 Ferrous ammonium citrate 50-100 Nicotinamide 1-10 L-Arginine hydrochloride 20-100 Pyridoxal hydrochloride 1-50 L-Cystine hydrochloride 10-500 Pyridoxine hydrochloride 1-10 L-Glutamine 150-600 Riboflavin 1-10 Glycine 10-80 Thiamine hydrochloride 1-10 L-Histidine hydrochloride 300-1500 Thymidine 0.1-1 L-Isoleucine 10-100 Vitamin E 1-10 L-Leucine 10-150 Vitamin B12 0.1-1 L-Lysine hydrochloride 20-150 Triiodothyronine (T3) 0.3-3 L-Methionine 50-250 Hydrocortisone 0.1-1 L-Phenylalanine 50-150 Recombinant human insulin 0.001-0.01 L-Serine 20-150 Ethanolamine 0.1-10 L-Threonine 20-250 Hypoxanthine 2-50 L-Alanine 50-150 EDTA 1-10 L-Asparagine 50-200 Glutathione 2-20 L-Aspartate 1-100 EGF 0.01-0.1 L-Cysteine hydrochloride 10-100 Cholesterol 0.5-3 L-Glutamic acid 10-100 L-Proline 0.001-0.01 Dextran sulfate 20-150 L-Tryptophan 1-10 Sodium bicarbonate 5-60 F68 500-2000 Figure 1 20-200 Figure 2 10-100 Figure 3 20-200 Figure 4 1500-3500

[0041] The present application further provides the use of the serum-free culture medium for Vero cells in domesticating Vero adherent cells into serum-free suspension cells.

[0042] The present application further provides a method for domesticating Vero adherent cells into serum-free suspension cells, which comprises the following steps:

[0043] (1) single-layer Vero adherent cells are digested and dispersed, and then adherent cultured in a first serum-free medium for 1-5 generations;

[0044] (2) the cells obtained after step (1) are digested and collected, and then suspended cultured in a culture solution containing a second serum-free medium and an anti-cell clumping agent for 3-5 generations, and then suspended cultured in the second serum-free medium to obtain domesticated Vero cells, wherein the second serum-free medium is a Vero cell serum-free medium according to an embodiment of the present application.

[0045] In some alternative embodiments, the first serum-free medium in step (1) is VP-SFM serum-free medium.

[0046] In some alternative embodiments, the adherent culture in step (1) is performed in a culture flask, and the inoculation density of the culture flask is (1-10) × 10 6 cells / T75 flask, and the culture temperature of the culture flask is controlled at 37 ± 1℃.

[0047] In some alternative embodiments, the digestion in step (1) is performed by using a recombinant trypsin digestion solution.

[0048] In some alternative embodiments, the suspension culture in step (2) is performed in a shake flask, and the inoculation density of the shake flask is (0.3-1.2) × 10 6 cells / mL, the rotation speed of the shake flask is controlled at 130 ± 5 rpm, and the temperature is controlled at 37 ± 1℃.

[0049] In some alternative embodiments, the domesticated Vero cells in step (2) are cryopreserved to establish a cell bank.

[0050] In some alternative embodiments, the cryopreservation solution used for cryopreservation contains a mixture of the serum-free medium according to an embodiment of the present application and DMSO, and the cell density is adjusted to (1-3) × 10 6 cells / mL.

[0051] The present application further provides use of the aforementioned Vero cell serum-free medium in amplifying viruses.

[0052] In some alternative embodiments, the viruses are sensitive viruses to Vero cells, including but not limited to rabies virus, poliovirus, coronavirus, rotavirus, influenza virus, porcine epidemic diarrhea virus, pseudorabies virus, and other sensitive viruses.

[0053] The Vero cell serum-free culture medium and the domesticated Vero cells thereof can be used to carry out cell suspension culture for virus culture and preparation, and the digestion operation is not needed in the process of cell passage to realize linear amplification of culture scale, and the cell density can reach 10 7 / mL, which lays a foundation for upgrading of corresponding virus vaccine production process.

[0054] The Vero cells are cultured in serum-free suspension in a shake flask or a bioreactor, and when the cell density reaches 1×10 6 / mL or above, the virus culture can be carried out by means of liquid exchange (after the cells are settled or intercepted, fresh serum-free culture solution is added) or direct inoculation of the virus. 50 The sensitive virus is inoculated according to M.O.I (multiplicity of infection) 0.0001-10, the culture temperature is controlled at 30-37.5℃, the pH is controlled at 6.8-7.5, the DO (dissolved oxygen) is controlled at 20-80%, the rotation speed is controlled at 30-200rpm, and the culture is maintained for a period of time, and the virus titer (such as TCID 6 / mL), antigen content (ug / mL) and the like are detected, the virus is harvested in time when the peak value is reached, and the harvested virus can be subjected to downstream purification, vaccine preparation and the like.

[0055] Some embodiments of the present application also provide a method for amplifying virus, comprising the following steps:

[0056] The domesticated Vero cells are subjected to suspension culture amplification by using the Vero cell serum-free culture medium of the foregoing embodiments of the present application, and then the virus is directly inoculated for culture.

[0057] In some optional embodiments, when the cell density in the cell culture solution reaches (1-5)×10 6 / mL or above, the virus is inoculated according to M.O.I 0.001-1, the culture temperature is controlled at 37±1℃, the pH is controlled at 7-7.5, the DO is controlled at 20-60%, and the rotation speed is controlled at 100-150rpm.

[0058] In some optional embodiments, the virus includes but is not limited to porcine pseudorabies virus, rabies virus, poliovirus, coronavirus, rotavirus, influenza virus, porcine epidemic diarrhea virus and other sensitive viruses.

[0059] In the present application, the term multiplicity of infection (M.O.I.) refers to the ratio of the number of viruses to the number of cells in the study of virus infection of cells.

[0060] In this document, HEPES represents 4-hydroxyethylpiperazine ethanesulfonic acid; EDTA represents ethylenediaminetetraacetic acid; EGF represents epidermal growth factor; F68 represents Poloxamer, CAS: 9003-11-6.

[0061] The above scheme is further described in connection with specific examples. It should be understood that the examples are used to illustrate the basic principles, main features and advantages of the present application, and the present application is not limited in scope by the examples below; the implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not noted are usually the conditions in conventional experiments.

[0062] Example 1

[0063] The present example provides a serum-free culture medium for Vero cells, which is composed of the following components:

[0064]

[0065]

[0066] The cell density cultured by using the serum-free culture medium for Vero cells can reach more than 10 7 / mL, the cell viability is more than 90%, the cells are well dispersed, and the cells can still grow and proliferate normally after 20 generations of continuous subculture.

[0067] Example 2

[0068] The present example provides domestication of Vero adherent cells into serum-free suspension cells and construction of a cell bank

[0069] 1. Materials used

[0070] Vero cells (ATCC, CCL-81), VP-SFM serum-free culture medium (source of culture, product number H513KJ), recombinant trypsin digestion solution (source of culture, product number S342KJ), anti-cell clumping agent (source of culture, product number S490JV), serum-free culture medium of Example 1, Dulbecco's phosphate buffer (DPBS, source of culture, product number B210KJ), T75 cell culture flask (Corning), 125 mL shake flask (Corning), centrifuge tube (Corning), DMSO (Sigma), 2 mL cryopreservation tube (Corning), benchtop low-speed centrifuge, cell cryopreservation box (CKSLAB), etc.

[0071] 2. Serum-free adaptation culture of adherent cells

[0072] 2.1 Under a microscope, select Vero adherent cells in the middle of the logarithmic growth phase and in a good monolayer in a T75 culture flask as the adaptation culture, and record the cell passage as P0;

[0073] 2.2 Discard the original culture supernatant, wash the cells once with DPBS solution, add 3 mL of recombinant trypsin digestion solution, digest at 37°C for 7 min, discard the digestion solution, add 10 mL of VP-SFM serum-free medium, and transfer the dispersed cells to a centrifuge tube by blowing, centrifuge at 1000 rpm for 3 min;

[0074] 2.3 Resuspend the cells with VP-SFM serum-free medium, inoculate 4×10 6 cells / mL into a T75 bottle and incubate at 37°C, 5% CO2;

[0075] 2.4 Incubate for 2-3 days until the cells are fully grown, and then continuously subculture for 2 generations according to the above method. After the cells stably proliferate, prepare for suspension adaptation culture.

[0076] 3. Vero cell serum-free suspension adaptation culture

[0077] 3.1 After the cells are cultured for 2 generations in VP-SFM medium without serum, collect the cells by digestion, inoculate 1×10 6 cells / mL into a 125 mL shake flask, and incubate. The cell passage is recorded as P3, the culture medium is serum-free medium of Example 1 + 5% anti-cell clumping agent, 30 mL / flask, and the shaking speed is controlled at 130 rpm, 37°C, 5% CO2;

[0078] 3.2 Sample and count every 2-3 days, and continuously subculture at a density of 1×10 6 cells / mL;

[0079] 3.3 After 5 generations of continuous subculture, when the cell density proliferates to more than 5×10 6 cells / mL, remove the anti-cell clumping agent and only use the serum-free medium of Example 1. The cell passage is recorded as P8;

[0080] 3.4 After 2 generations of continuous subculture, the cell passage is recorded as P10, and the cells are prepared for freezing to establish a serum-free Vero suspension cell bank.

[0081] 4. Serum-free Vero suspension cell freezing and bank establishment

[0082] 4.1 Centrifuge the serum-free Vero suspension cells (P10) at 1000 rpm for 3 min to collect them. The serum-free Vero suspension cells are the domesticated Vero suspension cells;

[0083] 4.2 Discard the supernatant, resuspend the cells with cell freezing solution (serum-free medium of Example 1 : DMSO = 9:1), adjust the cell density to 2×10 7 cells / mL, and divide the cell suspension into cryotubes, 1 mL / tube;

[0084] 4.3 Put the cryopreservation tube containing the cell suspension into the cryopreservation box, freeze in the ultra-low temperature refrigerator at -80°C, and take out the cells after overnight and store in liquid nitrogen.

[0085] Results: As shown in Figure 5 , the Vero suspension cells obtained after domestication appeared as single round cells in suspension under a microscope, without clumping and adherent cells.

[0086] ​ The growth of the Vero suspension cells obtained after domestication using the serum-free medium of Example 1 is shown. The culture conditions were 37°C, 5% CO2, and a rotation speed of 130 rpm.

[0087] ​ The growth of the Vero cells obtained after domestication using the serum-free medium of Example 1 after multiple generations of subculture is shown. The culture conditions were 37°C, 5% CO2, and a rotation speed of 130 rpm. The cells could still grow and proliferate normally after 20 generations of continuous subculture.

[0088] Example 3

[0089] This example provides a method for amplifying porcine pseudorabies virus (PRV)

[0090] 1. Materials used

[0091] Vero suspension cells of Example 2, serum-free medium of Example 1, shake flask (250 mL, Corning), bioreactor (10 L, Chu Yi), bioreactor (100 L, Chu Yi), porcine pseudorabies virus (PRV), low-speed centrifuge, carbon dioxide constant temperature shaking incubator, inverted phase contrast microscope, clean bench, etc.

[0092] 2. Suspension culture amplification of Vero serum-free suspension cells

[0093] Take one cryopreserved Vero suspension cell from the liquid nitrogen tank, shake it in 37°C warm water to quickly melt it, and transfer it aseptically to a 10 mL centrifuge tube. Centrifuge at 1000 rpm for 3 min to remove the supernatant, resuspend the cells with the serum-free medium of Example 1, and adjust the cell density to 0.5 x 10 6 / mL for inoculation in a 250 mL shake flask with a culture volume of 100 mL. Place it in a constant temperature shaking incubator and set the culture temperature to 37°C, 5% CO2, and a rotation speed of 130 rpm. The cells grow for about 2 days, and then continuously subculture and amplify at an inoculation density of 0.5 x 10 6 / mL.

[0094] Concentrate the cell suspension and aseptically transfer it to a 10 L bioreactor with a cell inoculation density of 0.5 x 106 / mL, culture volume 8L, reactor temperature 37℃, pH 7.0, DO 60%, stirring speed 80rpm, when the cell density reached 5x10 6 / mL, the cell suspension was transferred to a 100L bioreactor, cell inoculation density 0.5x10 6 / mL, culture volume 80L, reactor temperature 37℃, pH 6.9, DO 50%, stirring speed 120rpm.

[0095] 3. Serum-free suspension culture of PRV in bioreactor

[0096] When the cell density in the 100L bioreactor reached 5x10 6 / mL, the bioreactor temperature was controlled at 37℃, the pH was adjusted to 7.3, DO 40%, stirring speed 120rpm, and the culture was continued for 2 days at M.O.I of 0.01 porcine pseudorabies virus (PRV), and the virus TCID 50 was determined by sampling every 6h, and the virus was harvested and stored at 4℃, and subsequent cell debris removal, purification, inactivation and other treatments were performed.

[0097] Results: ​ The inverted microscope photographs of Vero cells showing lesions after inoculation of porcine pseudorabies virus are shown.

[0098] ​ The relationship between the time of suspension culture of Vero cells showing lesions after inoculation of porcine pseudorabies virus and the virus titer is shown, and the virus titer LgTCID 50 / mL reached above 6.8 after 30h of culture.

[0099] It should be noted that "source" in "source" in the "source" in the above examples refers to Shanghai Source Biotech Co., Ltd.

[0100] The above embodiments are only used to illustrate the embodiments of the present application, and are not intended to limit the embodiments of the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the embodiments of the present application, therefore all equivalent technical solutions also belong to the scope of the embodiments of the present application, and the patent protection scope of the embodiments of the present application should be defined by the claims.

Claims

1. A serum-free culture medium for Vero cells, characterized in that, The serum-free culture medium comprises the following components: 。 2. The serum-free medium for Vero cells according to claim 1, characterized in that, The serum-free culture medium comprises the following components:

3. The serum-free medium for Vero cells according to claim 1, characterized in that, The serum-free culture medium comprises the following components:

4. Use of the serum-free culture medium for Vero cells according to any one of claims 1-3 in domesticating Vero adherent cells into serum-free suspension cells.

5. A method for domesticating Vero adherent cells into serum-free suspension cells, characterized in that, The method comprises the following steps: (1) dispersing monolayer Vero adherent cells, and adherent culturing in a first serum-free culture medium for 1-5 generations; (2) collecting the cells obtained in step (1), and suspension culturing in a culture solution containing a second serum-free culture medium and an anti-cell clumping agent for 3-5 generations, and then suspension culturing in the second serum-free culture medium to obtain domesticated Vero cells, wherein the second serum-free culture medium is the serum-free culture medium according to any one of claims 1-3.

6. The method of taming Vero adherent cells to serum-free suspension cells according to claim 5, characterized in that, In step (1), the first serum-free medium is VP-SFM serum-free medium; and / or, In step (1), the adherent culture is performed in a culture flask, and the seeding density of the culture flask is (1-10) x 10 6 cells / T75 flask; and / or, In step (2), the suspension culture is carried out in a shake flask, and the inoculation density of the shake flask is (0.3-1.2) x 10 6 cells / mL, the rotation speed of the shake flask is controlled at 130±5 rpm, and the temperature is 37±1℃.

7. Use of the serum-free culture medium for Vero cells according to any one of claims 1-3 in amplifying viruses.

8. Use according to claim 7, characterized in that, The viruses are sensitive viruses for Vero cells, including rabies virus, poliovirus, coronavirus, rotavirus, influenza virus, porcine epidemic diarrhea virus, pseudorabies virus and other sensitive viruses.

9. A method of amplifying a virus, characterized by, The method comprises the following steps: The domesticated Vero cells are suspension cultured and amplified by using the serum-free culture medium for Vero cells according to any one of claims 1-3, and then viruses are directly inoculated for culturing.

10. The method of claim 9, wherein, When the cell density in the cell culture solution reaches (1-5) x 10 6 / mL or more, the virus is inoculated at M.O.I of 0.0001-10, and the culture temperature is controlled at 30-37.5°C, pH 6.8-7.5, DO 20-80%, and the rotation speed is 30-200 rpm; and / or, The viruses are porcine pseudorabies virus, rabies virus, poliovirus, coronavirus, rotavirus, influenza virus, porcine epidemic diarrhea virus and other sensitive viruses.

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