Culture medium for serum-free suspension culture of F81 cells and application of culture medium

Abstract

The invention relates to a culture medium for serum-free suspension culture of F81 cells and application of the culture medium, the culture medium comprises the following components in concentration: 4000-4500 mg / L of amino acid, 80-120 mg / L of vitamin, 5000-5100 mg / L of inorganic salt, 5000-5500 mg / L of glucose, 1000-1030 mg / L of adherence promoting substance, 20-40 mg / L of growth factor, 2000-2040 mg / L of buffering agent and 2-2.4 mg / L of antioxidant, the pH value of the culture medium is 7.0-7.2, and the pH value of the culture medium is 0.5-1.5. The antioxidant is prepared from partially hydroxylated glutathione and a carboxymethyl chitosan-gallic acid grafted copolymer. The partially hydroxylated glutathione and the carboxymethyl chitosan-gallic acid graft copolymer are compounded to serve as an antioxidant of the culture medium, so that the oxidation resistance of the culture medium is enhanced synergistically, and the cell survival rate is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological products, in particular to a culture medium for serum-free suspension culture of F81 cells and application thereof. BACKGROUND

[0002] F81 cells, as a cat kidney-derived epithelial cell line, have high sensitivity to feline panleukopenia virus (feline parvovirus), feline herpesvirus and other viruses, and have become the core host cell in the field of veterinary vaccine production. With the expansion of the industrialization scale of animal vaccines, the traditional adherent culture mode is gradually replaced by serum-free suspension culture technology due to low space utilization, complicated operation and easy contamination. Serum-free suspension culture can realize high-density large-scale culture of F81 cells, significantly improving the virus production efficiency, but the problem of oxidative stress damage faced by cells in this system has become a key bottleneck restricting the culture effect. During the serum-free suspension culture of F81 cells, the causes of oxidative stress are multi-source. On the one hand, the mechanical shear force of suspension culture can damage the integrity of the cell membrane, leading to an increase in the release of intracellular reactive oxygen species (ROS); on the other hand, the metabolic rate of cells increases during high-density culture, and a large amount of ROS such as superoxide anion and hydrogen peroxide produced by the mitochondrial respiratory chain accumulates, aggravating oxidative stress. Studies have shown that when the level of intracellular reactive oxygen species in F81 cells exceeds the physiological threshold, it will trigger lipid peroxidation, protein oxidation and DNA damage, ultimately leading to an increase in the apoptosis rate and growth arrest of cells, making it difficult to achieve large-scale culture of F81 cells. SUMMARY

[0003] In order to improve the cell survival rate of F81 cells, the present application provides a culture medium for serum-free suspension culture of F81 cells and application thereof.

[0004] In a first aspect, the present application provides a culture medium for serum-free suspension culture of F81 cells, which adopts the following technical solution: A culture medium for serum-free suspension culture of F81 cells, the culture medium comprises the following components in the following concentrations: 4000-4500 mg / L amino acids, 80-120 mg / L vitamins, 5000-5100 mg / L inorganic salts, 5000-5500 mg / L glucose, 1000-1030 mg / L adhesion-promoting substances, 20-40 mg / L growth factors, 2000-2040 mg / L buffering agents, and 2-2.4 mg / L antioxidants, the pH value of the culture medium is 7.0-7.2, and the antioxidants comprise partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer.

[0005] Amino acids are the core raw materials for cells to synthesize proteins in the components of the serum-free suspension culture medium, which are the basis for F81 cell growth, proliferation and repair of damaged structures; vitamins are involved in cell metabolism in the form of coenzymes or cofactors, which guarantee the balance between cell metabolism efficiency and physiological function; the role of inorganic salts is to maintain the osmotic pressure balance of the cultured cells, guarantee cell signal transduction and enzyme activity; glucose is the main energy source for F81 cells; the role of pro-adhesion substances is to reduce cell aggregation and maintain the activity of single cells in suspension state; the role of growth factors is to accelerate cell division and proliferation while inhibiting apoptosis signals in suspension culture; the core role of buffer is to stabilize the pH value of the culture medium in the appropriate range of 7.0-7.2; the role of antioxidants is to scavenge active oxygen produced by cell metabolism, reduce the apoptosis rate of cells, and maintain the activity and function of long-term cultured cells.

[0006] The inventors found that adding partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer as antioxidants of the culture medium unexpectedly improves the antioxidant performance of the serum-free suspension culture medium and also improves the cell survival rate of F81 cells cultured in the culture medium. The carboxyl and amino groups of carboxymethyl chitosan can chelate with transition metal ions such as iron ions and copper ions in the culture medium, which can reduce the formation of free radicals, and the high molecular chain of carboxymethyl chitosan can form a protective film on the surface of F81 cells during the culture of F81 cells, which can intercept extracellular active oxygen. The phenolic hydroxyl groups of gallic acid can react with DPPH free radicals, ABTS free radicals and other active oxygen to combine into harmless substances, thereby efficiently eliminating extracellular active oxygen. Therefore, carboxymethyl chitosan-gallic acid graft copolymer can improve the synergistic antioxidant effect of the two and improve the cell survival rate.

[0007] The sulfhydryl group on the partially hydroxylated glutathione is combined with active oxygen such as hydroxyl radicals, superoxide anions and the like in F81 cells by reaction, and is converted into harmless substances, thereby efficiently eliminating intracellular active oxygen. After the glutathione is modified by the hydroxyl group, the hydroxyl-carboxyl, hydroxyl-hydroxyl and other various bonds are formed with the carboxymethyl chitosan-gallic acid graft copolymer, thereby not only anchoring the glutathione on the polymer chain of the carboxymethyl chitosan-gallic acid graft copolymer through hydrogen bonds to improve the uniform distribution of the antioxidant component in the culture medium, and further improve the antioxidant effect; but also effectively maintain the antioxidant effect of the phenolic hydroxyl group. Due to the hydrogen bond-mediated combination of glutathione and carboxymethyl chitosan-gallic acid graft copolymer to form a proximity effect, when the antioxidant complex contacts the cells, the carboxymethyl chitosan-gallic acid graft copolymer first functions extracellularly, and the glutathione can be quickly taken up into the intracellular by the cells, forming a continuous antioxidant chain in space, covering the entire path of active oxygen generation, greatly improving the antioxidant performance of the antioxidant. The present application realizes the dual strong protection of extracellular interception and intracellular purification of F81 cells in the culture medium through the synergistic effect of partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer, greatly improving the cell survival rate of F81 cells in the culture medium.

[0008] In a specific implementable scheme, the concentration ratio of the above partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer is (5-15): 1.

[0009] The antioxidant prepared by the above ratio greatly improves the antioxidant defense performance of the serum-free suspension culture medium. If the content of partially hydroxylated glutathione is too high, the carboxymethyl chitosan-gallic acid graft copolymer will be relatively insufficient, which will weaken the extracellular antioxidant effect of the antioxidant and make it difficult to effectively cooperate with glutathione to form an efficient intracellular and extracellular antioxidant, resulting in incomplete elimination of extracellular active oxygen and easy cell damage, thereby reducing the cell survival rate. If the content of partially hydroxylated glutathione is too low, it cannot effectively eliminate the intracellular active oxygen, which will aggravate the intracellular oxidative stress, and the carboxymethyl chitosan-gallic acid graft copolymer can only function extracellularly, but lacks the cooperation of glutathione intracellularly, and cannot comprehensively cope with the oxidative stress inside and outside the cells, thereby improving the cell survival rate and proliferation efficiency.

[0010] In a specific implementable scheme, the preparation steps of the above partially hydroxylated glutathione are as follows: Dissolve the glutathione in distilled water, add ascorbic acid, then adjust the pH of the solution to 8.4-8.6, add epoxyl ethanol under nitrogen condition, and then treat to obtain partially hydroxylated glutathione.

[0011] The 50% of the proportion of the thiol group of the partially hydroxylated glutathione prepared by the above steps is modified, which not only retains the ability to scavenge intracellular free radicals, but also forms multi-dimensional hydrogen bonds with the carboxymethyl chitosan-gallic acid graft copolymer, realizes the anchoring and dispersion of the two, maximizes the retention of phenolic hydroxyl activity, improves the antioxidant effect, and makes the serum-free suspension medium have excellent antioxidant performance.

[0012] In a specific implementable embodiment, the preparation steps of the above carboxymethyl chitosan-gallic acid graft copolymer are as follows: O-carboxymethyl chitosan is dissolved in distilled water, ascorbic acid and gallic acid are added, and then the pH of the solution is adjusted to 5.0-7.0, hydrogen peroxide is added under nitrogen condition, and then the reaction is treated to obtain the carboxymethyl chitosan-gallic acid graft copolymer.

[0013] The carboxymethyl chitosan-gallic acid graft copolymer prepared by the above steps is used in the serum-free suspension medium in cooperation with the partially hydroxylated glutathione, so that the serum-free suspension medium has excellent antioxidant performance.

[0014] In a specific implementable embodiment, the above amino acids include the following components at the following concentrations: 400-600 mg / L L-alanyl-L-glutamine, 400-600 mg / L L-arginine hydrochloride, 40-60 mg / L L-cystine hydrochloride, 40-60 mg / L L-cysteine hydrochloride, 60-100 mg / L glycine, 60-100 mg / L L-histidine hydrochloride, 100-200 mg / L L-isoleucine, 100-200 mg / L L-leucine, 150-250 mg / L L-lysine hydrochloride, 80-120 mg / L L-methionine, 180-220 mg / L L-phenylalanine, 60-100 mg / L L-serine, 180-220 mg / L L-threonine, 60-100 mg / L L-tryptophan, 80-120 mg / L L-tyrosine, 80-120 mg / L L-valine, 1800-2200 mg / L yeast hydrolysate, and 8-12 mg / L ethanolamine.

[0015] By adopting the above technical solution, the amino acid components within the limited concentration range can provide sufficient and balanced nitrogen source and energy precursor for F81 cells, meet the metabolic needs such as protein synthesis during cell growth and proliferation. Among them, the reasonable collocation of various essential amino acids and non-essential amino acids can promote smooth cell metabolism, improve cell activity and growth rate, and provide basic nutritional guarantee for the good state of cells in a serum-free suspension environment.

[0016] In one specific embodiment, the vitamins include the following components at the following concentrations: 8-12 mg / L folic acid, 10-20 mg / L myo-inositol, 8-12 mg / L nicotinamide, 8-12 mg / L D-calcium pantothenate, 10-20 mg / L pyridoxal hydrochloride, 10-20 mg / L pyridoxine hydrochloride, 0.2-0.8 mg / L riboflavin, 10-20 mg / L thiamine hydrochloride.

[0017] By adopting the technical solution, according to the metabolic characteristics of F81 cells, the vitamin components in the concentration range can participate in various enzymatic reactions and metabolic regulation of the cells, so that the vitamin formula is more suitable for the needs of the cells, the efficiency of the vitamins in cell metabolism is improved, and more accurate vitamin nutritional support is provided for the cells.

[0018] In one specific embodiment, the inorganic salts include the following components at the following concentrations: 80-120 mg / L calcium chloride, 40-60 mg / L ferric nitrate nonahydrate, 40-60 mg / L magnesium sulfate, 100-200 mg / L potassium chloride, 1000-3000 mg / L sodium bicarbonate, 2000-4000 mg / L sodium chloride, 0.05-0.15 mg / L zinc sulfate heptahydrate, 40-60 mg / L sodium dihydrogen phosphate, 40-60 mg / L choline chloride.

[0019] By adopting the technical solution, according to the environmental characteristics of serum-free suspension culture of F81 cells, the inorganic salt components in the above-mentioned concentrations can maintain the appropriate osmotic pressure and ion balance of the culture medium, and provide the necessary mineral elements for the cells. In particular, the components such as sodium bicarbonate and sodium chloride that affect the osmotic pressure are precisely controlled, so that the inorganic salt system is more conducive to the growth of cells in a suspended state.

[0020] In one specific embodiment, the glucose includes the following components at the following concentrations: 4800-5200 mg / L D-glucose, 50-150 mg / L sodium pyruvate, 150-250 mg / L dextran sulfate.

[0021] By adopting the technical solution, the glucose component can provide sufficient energy source for F81 cells. D-glucose is the main energy source for cells, sodium pyruvate can be used as an emergency energy supplement, and dextran sulfate can regulate cell proliferation and adhesion. The synergistic effect of the three can ensure stable energy supply for cells during serum-free suspension culture, promote cell metabolism and proliferation, and improve cell density.

[0022] In one specific embodiment, the above-mentioned adhesion-promoting substance comprises the following components at the following concentrations: 10-20 mg / L transferrin, 800-1200 mg / L F68, and / or the above-mentioned growth factor comprises the following components at the following concentrations: 18-22 mg / L recombinant human insulin, 0.08-0.12 mg / L EGF, and 8-12 mg / L cholesterol, and / or the above-mentioned buffer comprises the following components at the following concentrations: 1000-3000 mg / L HEPES and 10-30 mg / L phenol red sodium.

[0023] By adopting the above technical solution, the adhesion-promoting substance in the concentration range can assist F81 cells in adapting to the suspension environment in serum-free suspension culture. Transferrin can promote iron ion absorption, and F68 can reduce the damage of shear force to cells. The two components can synergistically improve the tolerance of cells to suspension culture conditions, reduce cell aggregation and damage, and maintain a good suspension growth state of cells.

[0024] The growth factor component can effectively promote the growth and proliferation of F81 cells. Recombinant human insulin regulates cell metabolism and growth signals. EGF promotes cell proliferation, and cholesterol maintains cell membrane stability. The three components in the specific concentration range can synergistically activate cell growth-related pathways, improve cell proliferation rate, and increase cell density, thereby providing support for large-scale cell culture.

[0025] The buffer component can effectively maintain the pH value of the culture medium at 7.0-7.2. HEPES can buffer pH changes in a wide range, and phenol red sodium can directly indicate pH changes. The two components can synergistically resist the influence of acid-base substances produced by cell metabolism on the pH of the culture medium, thereby providing a stable acid-base environment for F81 cells and ensuring normal physiological functions of the cells.

[0026] In a second aspect, the application provides an application of a culture medium for serum-free suspension culture of F81 cells, which adopts the following technical solution: The application of the culture medium for serum-free suspension culture of F81 cells uses the culture medium for serum-free suspension culture of F81 cells of the first aspect to domesticate F81 cells, and controls the density of F81 cells in the culture medium to be 0.8-1.2×10 6 cells / mL.

[0027] By adopting the above technical solution, controlling the initial density of F81 cells to be 0.8-1.2×10 6 cells / mL can enable the cells to grow and proliferate efficiently in the culture medium. At this density, the cells can fully utilize the nutrients in the culture medium, reduce competition between cells and accumulation of metabolic waste, improve the growth rate and survival rate of the cells, and facilitate large-scale culture of F81 cells.

[0028] To sum up, the present application includes at least one of the following beneficial technical effects: 1、The present application uses partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer as antioxidants in the culture medium, which synergistically enhances the antioxidant performance of the culture medium and unexpectedly improves the cell survival rate of F81 cells cultured in the culture medium.

[0029] 2、The present application controls the compounding ratio of partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer, balances the oxidative stress inside and outside the cell, and synergistically realizes the double protection of extracellular interception and intracellular purification for F81 cells in the culture medium, thereby enhancing the antioxidant performance of the culture medium.

[0030] 3、The present application adds D-glucose, sodium pyruvate and dextran sulfate as glucose components into the culture medium, and the three components synergistically work to ensure stable energy supply for cells during serum-free suspension culture, promote cell metabolism and proliferation, and improve cell density. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 1 is a microscope image of F81 adherent cells recovered before culture; Figure 2 Figure 2 is a microscope image of F81 suspension cells cultured in a serum-free medium according to Example 1 of the present application; Figure 3 Figure 3 is a 6-day culture growth curve of F81 suspension cells cultured in a serum-free medium according to Example 1 of the present application; Figure 4 Figure 4 is a microscope image of F81 suspension cells cultured in a serum-free medium according to Example 1 of the present application, which is applied to the cell pathology after inoculation of feline parvovirus. DETAILED DESCRIPTION

[0032] The present application will be further described in detail below in combination with examples and comparative examples: Some of the raw materials used in the examples and comparative examples are as follows: Yeast hydrolysate (brand: Yeastolate™), F68 (brand: P188), transferrin (item number: 12389019), recombinant human insulin (item number: A11382II) are all purchased from Thermo Fisher Gibco™; F81 adherent cells are purchased from the American Type Culture Collection; calcium and magnesium-free PBS and DMEM culture medium are both from Tianxinhe (Suzhou) Biotechnology Co., Ltd.; Trpzyme®recombinant trypsin digestion solution is purchased from Gbico Company; newborn bovine serum is purchased from Lanzhou Rongye Biological Technology Co., Ltd.; T25 culture flask, T75 culture flask, T175 culture flask, 125 mL shake flask and 250 mL shake flask are all purchased from Corning Life Sciences Co., Ltd.

[0033] Preparation of hydroxylated glutathione: Dissolve 1.54 g of glutathione in 100 mL of distilled water, add 10 mg of ascorbic acid, adjust the pH of the solution to 8.5 with 0.1 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and add 0.12 g of epoxy ethanol under nitrogen at room temperature for 2.5 h. After the reaction is completed, the reaction mixture is placed in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed with distilled water for 18 h. Finally, the dialysate is freeze-dried to obtain hydroxylated glutathione.

[0034] Preparation of carboxymethyl chitosan-gallic acid graft copolymer: Dissolve 0.5 g of O-carboxymethyl chitosan in 50 mL of distilled water, add 1.32 g of ascorbic acid and 0.5 g of gallic acid, adjust the pH of the solution to 6.0 with 0.1 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, and add 0.375 mL of 10 mol / L H2O2 solution under nitrogen at room temperature for 24 h. After the reaction is completed, the reaction mixture is placed in a dialysis bag with a molecular weight cut-off of 3000 Da, and dialyzed with distilled water for 72 h. Finally, the dialysate is freeze-dried to obtain carboxymethyl chitosan-gallic acid graft copolymer.

[0035] The related raw materials used in the examples and comparative examples are not specified, and are all conventional products that can be purchased in the market.

[0036] Example Example 1 A method for preparing a culture medium for serum-free suspension culture of F81 cells: The amino acids, vitamins, inorganic salts, glucose, pro-adhesion substances, growth factors, buffers and antioxidants that make up the serum-free cell culture medium are obtained by weighing according to the formula, pre-mixing, crushing, final mixing, and sub-packaging steps to obtain serum-free medium dry powder; S1-1, for example, 1L of medium preparation, weigh 18277.9 mg of serum-free medium dry powder, and take 1L of ultrapure water. Add the serum-free medium dry powder to the water and stir to dissolve thoroughly; S2-1, add 2 g / L of sodium bicarbonate and adjust the pH to 7.1 with sodium hydroxide S3-1, filter sterilization using a 0.22 um filter membrane under positive pressure; S4-1, store the sterilized liquid medium at 5°C.

[0037] The composition of 1L of serum-free cell culture medium in this example contains the following specific component concentrations: A1, the total concentration of amino acid components is 4360 mg / L, including the following specific concentration components: 500 mg / L L-alanyl-L-glutamine, 500 mg / L L-arginine hydrochloride, 50 mg / L L-cystine hydrochloride, 50 mg / L L-cysteine hydrochloride, 80 mg / L glycine, 80 mg / L L-histidine hydrochloride, 150 mg / L L-isoleucine, 150 mg / L L-leucine, 200 mg / L L-lysine hydrochloride, 100 mg / L L-methionine, 200 mg / L L-phenylalanine, 80 mg / L L-serine, 200 mg / L L-threonine, 80 mg / L L-tryptophan, 100 mg / L L-tyrosine, 100 mg / L L-valine, 2000 mg / L yeast hydrolysate, 10 mg / L ethanolamine A2, the total concentration of vitamin components is 100.5 mg / L, including the following specific concentration components: 10 mg / L folic acid, 15 mg / L myo-inositol, 10 mg / L nicotinamide, 10 mg / L D-calcium pantothenate, 15 mg / L pyridoxal hydrochloride, 15 mg / L pyridoxine hydrochloride, 0.5 mg / L riboflavin, 15 mg / L thiamine hydrochloride.

[0038] A3, the total concentration of inorganic salt components is 5450.1 mg / L, including the following specific concentration components: 100 mg / L calcium chloride, 50 mg / L ferric nitrate nonahydrate, 50 mg / L magnesium sulfate, 150 mg / L potassium chloride, 2000 mg / L sodium bicarbonate, 3000 mg / L sodium chloride, 0.1 mg / L zinc sulfate heptahydrate, 50 mg / L sodium dihydrogen phosphate, 50 mg / L choline chloride.

[0039] A4, the total concentration of glucose components is 5300 mg / L, including the following specific concentration components: 5000 mg / L D-glucose, 100 mg / L sodium pyruvate, 200 mg / L dextran sulfate.

[0040] A5, the total concentration of pro-adhesion substance components is 1015 mg / L, including the following specific concentration components: 15 mg / L transferrin, 1000 mg / L F68.

[0041] A6, the total concentration of growth factor components is 30.1 mg / L, including the following specific concentration components: 20 mg / L recombinant human insulin, 0.1 mg / L EGF, and 10 mg / L cholesterol.

[0042] A7, the total concentration of buffer components is 2020 mg / L, including the following specific concentration components: 2000 mg / L HEPES and 20 mg / L phenol red sodium.

[0043] A8, total antioxidant component concentration is 2.2 mg / L, including the following specific concentration components: 2 mg / L of partially hydroxylated glutathione, 0.2 mg / L of carboxymethyl chitosan-gallic acid graft copolymer.

[0044] The process of domesticating F81 cells by the above medium: S1-2, resuscitation of cells: take one F81 adherent cell cryopreservation tube from the liquid nitrogen tank, shake it in warm water at 37°C to quickly melt it, and transfer it to a 10 mL centrifuge tube under sterile conditions. Centrifuge at 1000 rpm for 5 min to remove the supernatant, resuspend with DMEM + 10% NBS, and inoculate the cells into a T25 culture flask. Incubate at 37°C with 5% CO2, and the adherent cell morphology is as shown in Figure 1 The cells are spindle-shaped and have a full shape. S2-2, cell subculture: after the cells form 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 are dispersed, and terminate the digestion with DMEM + 10% NBS. Subculture at a ratio of 1:3, and subculture the F81 cells 3 times according to the same method.

[0045] S3-2, cell serum-free adherent culture: the adherent F81 cells obtained above are cultured with DMEM medium containing 5%, 2%, 1%, and 0.5% newborn calf serum, respectively, and subcultured at a ratio of 1:3 in a 37°C, 5% CO2 cell incubator. The medium containing 5% and 2% newborn calf serum is subcultured for 2 generations each, and the medium containing 1% and 0.5% newborn calf serum is subcultured for 3 generations each, so that the cells gradually adapt to low serum adherent culture.

[0046] S4-2, serum-free suspension culture of cells: adjust the cell density of the last generation obtained in step S3-2 to 1×10 6 cells / mL with serum-free whole suspension culture medium, and culture at 37°C with 5% CO2 and 140 r / min shaking. Centrifuge and replace the medium for subculture without division until the cell density reaches 3×10 6 cells / mL. Adjust the cell density to 5×10 5 cells / mL with serum-free whole suspension culture medium, and culture at 37°C with 5% CO2 and 140 r / min shaking. After 10-20 generations of subculture, when the cells proliferate stably and there is no cell aggregation, the serum-free whole suspension culture type F81 cell line is domesticated and named F81-S.

[0047] S5-2, cell freezing and library construction: obtain stable suspension cells growing in serum-free suspension medium. The culture was centrifuged at 1000 rpm for 3 min to collect the cells, resuspended with F81 cell serum-free medium + 7% DMSO, and the cell density was adjusted to 3 x 10 7 cells / mL, aliquot the freezing tube to 80°C ultra-low temperature refrigerator overnight and then transfer to liquid nitrogen tank for long-term preservation.

[0048] As shown in Figure 2 , the F81 serum-free suspension cells obtained by domestication through the above process appear as single round cells under an inverted microscope, no clumping phenomenon, no adherent cells.

[0049] Example 2 Example 2 differs from Example 1 only in that the concentration of partially hydroxylated glutathione in A8 is replaced by 1.1 mg / L, and the concentration of carboxymethyl chitosan-gallic acid graft copolymer is replaced by 1.1 mg / L.

[0050] Example 3 Example 3 differs from Example 1 only in that the concentration of partially hydroxylated glutathione in A8 is replaced by 2.1 mg / L, and the concentration of carboxymethyl chitosan-gallic acid graft copolymer is replaced by 0.1 mg / L.

[0051] Comparative Example Comparative Example 1 Comparative Example 1 differs from Example 1 in that the partially hydroxylated glutathione in A8 is replaced by glutathione.

[0052] Comparative Example 2 Comparative Example 2 differs from Example 1 in that no carboxymethyl chitosan-gallic acid graft copolymer is added in A8, and the concentration of partially hydroxylated glutathione is replaced by 2.2 mg / L.

[0053] Comparative Example 3 Comparative Example 3 is based on Example 1, and Comparative Example 3 differs from Example 1 in that no partially hydroxylated glutathione is added in A8, and the concentration of carboxymethyl chitosan-gallic acid graft copolymer is replaced by 2.2 mg / L.

[0054] Performance detection test The serum-free suspension culture type F81 cells domesticated in Examples 1-3 and Comparative Examples 1-3 were adjusted to 1.0 x 10 6Cells / ml were seeded into a 50L bioreactor, with the following reactor parameters set: temperature 37℃, rotation speed 90rpm / min, dissolved oxygen 60%, and pH 7.0. Cells were sampled and counted every 24 hours to check cell viability.

[0055] Table 1 shows the cell density and cell viability of serum-free suspension culture F81 cells cultured for 6 days in Examples 1-3.

[0056] Table 1

[0057] Table 2 shows the cell density and cell viability of serum-free suspension cultured F81 cells cultured for 6 days in Comparative Examples 1-3.

[0058] Table 2

[0059] refer to Figure 2 , Figure 3 As shown in Table 1, the F81 suspension cells acclimated to the culture medium with the component concentration of Example 1 appeared as single, round, suspended cells under an inverted microscope, without clumping or adherent cells. Furthermore, the F81 suspension cells acclimated to the culture medium with the component concentration of Example 1 were cultured at a concentration of 1×10⁻⁶. 6 F81 suspension cells were seeded at a density of 5.8 × 10⁶ cells / ml and grew for 3 days to reach a density of 5.8 × 10⁶ cells / ml. 6 The cells / ml were well-formed, and the viability reached 99%, indicating that the F81 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 F81 cells.

[0060] Referring to Examples 1-3 and Table 1, it can be seen that the F81 suspension cells domesticated in Example 1 showed better growth, cell density increase, and cell viability over 6 days than those in Examples 2 and 3.

[0061] Referring to Example 1, Comparative Examples 1-3, and Tables 1 and 2, it can be seen that the F81 suspension cells acclimated to the culture medium with the component concentration of Example 1 showed better cell density proliferation and cell viability than Comparative Examples 1, 2, and 3 over 6 days. This may be because the glutathione in Comparative Example 1 was not hydroxylated, which is detrimental to its synergistic antioxidant effect with the carboxymethyl chitosan-gallic acid graft copolymer. Comparative Example 2 lacked the extracellular antioxidant capacity to scavenge reactive oxygen species due to the absence of the carboxymethyl chitosan-gallic acid graft copolymer, while Comparative Example 3 lacked the intracellular antioxidant capacity to scavenge reactive oxygen species due to the absence of partially hydroxylated glutathione. These factors all contribute to the poor proliferation density and cell viability of the F81 suspension cells acclimated to the culture medium.

[0062] The application adopts F81 cell serum-free whole suspension culture medium, so that the cells are in a high-density and high-viability proliferation state in suspension, avoids the operation process of adherent cell digestion, and easily realizes large-scale culture of F81 cells.

[0063] After the serum-free whole suspension culture type F81 cells of the culture medium of Example 1 were acclimated in suspension and grew in a 50L volume bioreactor for 3 days, part of the cells were transferred to the serum-free suspension culture medium prepared in Example 1, and the cell density was adjusted to 1x10 6 cells / mL, and the cells were inoculated with feline panleukopenia virus at an MOI of 0.8, and then cultured in a 37℃, 5% CO2 shaker. When the cell viability was less than 20%, the cells were harvested, and the cytopathic effect was observed. As shown in FIG. 2, there were many cell fragments with different sizes and shapes. The virus liquid after harvesting was repeatedly frozen and thawed three times at-80℃, and then centrifuged at 5000 rpm for 10 min. The supernatant virus liquid was collected and detected. Figure 4

[0064] After TCID 50 detection, the virus titer was 107.6 TCID 50 / 0.1mL. It can be seen that the serum-free whole suspension culture type F81 cell line acclimated in the culture medium of Example 1 of the application is sensitive to feline panleukopenia virus, which proves that the serum-free suspension culture medium of F81 prepared in the application has good application prospects in the production of feline panleukopenia virus.

[0065] This embodiment is only an explanation of the application, and is not a limitation of the application. Those skilled in the art can make modifications to this embodiment without creative contribution after reading this specification, but as long as the modifications are within the scope of the claims of the application, they are protected by the Patent Law.​

Claims

1. A culture medium for serum-free suspension culture of F81 cells, characterized in that, The culture medium comprises the following components at the following concentrations: 4000-4500 mg / L amino acids, 80-120 mg / L vitamins, 5000-5100 mg / L inorganic salts, 5000-5500 mg / L glucose, 1000-1030 mg / L adhesion promoters, 20-40 mg / L growth factors, 2000-2040 mg / L buffer, and 2-2.4 mg / L antioxidants. The pH of the culture medium is 7.0-7.

2. The antioxidants include partially hydroxylated glutathione and carboxymethyl chitosan-gallic acid graft copolymer.

2. The culture medium according to claim 1, characterized in that, The concentration ratio of the partially hydroxylated glutathione and the carboxymethyl chitosan-gallic acid graft copolymer is (5-15):

1.

3. The culture medium according to claim 1, characterized in that, The preparation steps of the partially hydroxylated glutathione are as follows: Glutathione was dissolved in distilled water, ascorbic acid was added, and the pH of the solution was adjusted to 8.4-8.

6. Epoxyethanol was added under nitrogen conditions to react, and post-treatment yielded partially hydroxylated glutathione.

4. The culture medium according to claim 1, characterized in that, The preparation steps of the carboxymethyl chitosan-gallic acid graft copolymer are as follows: O-Carboxymethyl chitosan was dissolved in distilled water, and ascorbic acid and gallic acid were added. The pH of the solution was then adjusted to 5.0-7.

0. Hydrogen peroxide was added under nitrogen conditions to react, and the post-treatment yielded a carboxymethyl chitosan-gallic acid graft copolymer.

5. The culture medium according to claim 1, characterized in that, The amino acids comprise the following components at the following concentrations: 400-600 mg / L L-alanyl-L-glutamine, 400-600 mg / L L-arginine hydrochloride, 40-60 mg / L L-cysteine ​​hydrochloride, 40-60 mg / L L-cysteine ​​hydrochloride, 60-100 mg / L glycine, 60-100 mg / L L-histidine hydrochloride, 100-200 mg / L L-isoleucine, 100-200 mg / L L-leucine, 150-250 mg / L L-lysine hydrochloride, 80-120 mg / L L-methionine, 180-220 mg / L L-phenylalanine, 60-100 mg / L L-serine, 180-220 mg / L L-threonine, 60-100 mg / L L-tryptophan, and 80-120 mg / L L-methionine. L-tyrosine, 80-120 mg / L L-valine, 1800-2200 mg / L yeast hydrolysate, 8-12 mg / L ethanolamine.

6. The culture medium according to claim 1, characterized in that, The vitamins comprise the following components at the following concentrations: 8-12 mg / L folic acid, 10-20 mg / L inositol, 8-12 mg / L nicotinamide, 8-12 mg / L D-calcium pantothenate, 10-20 mg / L pyridoxal hydrochloride, 10-20 mg / L pyridoxine hydrochloride, 0.2-0.8 mg / L riboflavin, and 10-20 mg / L thiamine hydrochloride.

7. The culture medium according to claim 1, characterized in that, The inorganic salts comprise the following components at the following concentrations: 80-120 mg / L calcium chloride, 40-60 mg / L ferric nitrate nonahydrate, 40-60 mg / L magnesium sulfate, 100-200 mg / L potassium chloride, 1000-3000 mg / L sodium bicarbonate, 2000-4000 mg / L sodium chloride, 0.05-0.15 mg / L zinc sulfate heptahydrate, 40-60 mg / L sodium dihydrogen phosphate, and 40-60 mg / L choline chloride.

8. The culture medium according to claim 1, characterized in that, The glucose comprises the following components at the following concentrations: 4800-5200 mg / L D-glucose, 50-150 mg / L sodium pyruvate, and 150-250 mg / L dextran sulfate.

9. The culture medium according to claim 1, characterized in that, The adhesion-promoting substance comprises the following components at concentrations: 10-20 mg / L transferrin, 800-1200 mg / L F68, and / or the growth factor comprises the following components at concentrations: 18-22 mg / L recombinant human insulin, 0.08-0.12 mg / L EGF and 8-12 mg / L cholesterol, and / or the buffer comprises the following components at concentrations: 1000-3000 mg / L HEPES and 10-30 mg / L sodium phenolate.

10. An application of a culture medium for serum-free suspension culture of F81 cells, characterized in that, F81 cells were acclimatized using the culture medium for serum-free suspension culture as described in any one of claims 1-9, with the density of F81 cells in the culture medium controlled at 0.8-1.2 × 10⁻⁶. 6 cells / mL.

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